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Arthrobotrys flagrans, a nematode‐eating fungus, is an effective component of animal parasitic nematode biocontrol agents. In the dried formulation, the majority of spores are in an endogenous dormant state. This study focuses on dormant chlamydospore and nondormant chlamydospore of A. flagrans to investigate the differences in cyclic adenosine monophosphate (cAMP) and protein content between the two types of spores. cAMP and soluble proteins were extracted from the nondormant chlamydospore and dormant chlamydospore of two isolates of A. flagrans. The cAMP Direct Immunoassay Kit and Bradford protein concentration assay kit (Coomassie brilliant blue method) were used to detect the cAMP and protein content in two types of spores. Results showed that the content of cAMP in dormant spores of both isolates was significantly higher than that in nondormant spores (p < 0.05). The protein content of dormant spores in DH055 bacteria was significantly higher than that of nondormant spores (p < 0.05). In addition, the protein content of dormant spores of the SDH035 strain was slightly higher than that of nondormant spores, but the difference was not significant (p > 0.05). The results obtained in this study provide evidence for the biochemical mechanism of chlamydospore dormancy or the germination of the nematophagous fungus A. flagrans. [ABSTRACT FROM AUTHOR]

Aims Chlamydospores are a special, differentiated type with high environmental resistance. Consequently, the chlamydospores of Trichoderma harzianum T4 can used to industrialize the latter. This study aimed to investigate the key factors affecting the sporulation type of T. harzianum T4 and the mechanisms underlying this effect. Methods and results In the liquid fermentation of T. harzianum T4, ammonium sulfate (AS) inhibited conidia formation and chlamydospore production. Fermentation tests revealed that acid stress induced sporulation type alteration. Transcriptomic analysis was used to evaluate the adaptation strategy and mechanism underlying spore type alteration under acid stress. The fermentation experiments involving the addition of amino acids revealed that branched-chain amino acids benefited conidia production, whereas β-alanine benefited chlamydospore production. Confocal microscope fluorescence imaging and chloroquine intervention demonstrated that vacuole function was closely related to chlamydospore production. Conclusion The sporulation type of T. harzianum T4 can be controlled by adjusting the fermentation pH. T. harzianum T4 cells employ various self-protection measures against strong acid stress, including regulating their metabolism to produce a large number of chlamydospores for survival. [ABSTRACT FROM AUTHOR]

ABSTRACT Arthrobotrys flagrans (Duddingtonia flagrans) is a typical nematode-trapping fungus which has been used for nematode biocontrol. The global regulator LaeA is widely distributed in filamentous fungi and plays a crucial role in secondary metabolism and development in addition to pathogenicity in fungal pathogens. In this study, the chromosome-level genome of A. flagrans CBS 565.50 was sequenced and homologous sequences of LaeA were identified in A. flagrans. A. flagrans LaeA (AfLaeA) knockout resulted in slower hyphal growth and a smoother hyphal surface. Importantly, deletion of AfLaeA resulted in the absence of chlamydospores and attenuated glycogen and lipid accumulation in hyphae. Similarly, disruption of the AfLaeA gene led to fewer traps and electron-dense bodies, lower protease activity, and a delay in capturing nematodes. The AfLaeA gene had a large effect on the secondary metabolism of A. flagrans, and both the deletion and overexpression of AfLaeA could yield new compounds, whereas some compounds were lost due to the absence of the AfLaeA. Protein-protein interactions between AfLaeA and another eight proteins were detected. Furthermore, transcriptome data analysis showed that 17.77% and 35.51% of the genes were influenced by the AfLaeA gene on days 3 and 7, respectively. AfLaeA gene deletion resulted in the higher expression level of the artA gene cluster, and multiple differentially expressed genes involved in glycogen and lipid synthesis and metabolism showed opposite expression patterns in wild-type and ΔAfLaeA strains. In summary, our results provide novel insights into the functions of AfLaeA in mycelial growth, chlamydospore production, pathogenicity, secondary metabolism, and energy metabolism in A. flagrans. IMPORTANCE The regulation of biological functions, such as the secondary metabolism, development, and pathogenicity of LaeA, has been reported in multiple fungi. But to date, no study on LaeA in nematode-trapping fungi has been reported. Moreover, it has not been investigated whether or not LaeA is involved in energy metabolism and chlamydospore formation has not been investigated. Especially in the formation mechanism of chlamydospores, several transcription factors and signaling pathways are involved in the production of chlamydospores, but the mechanism of chlamydospore formation from an epigenetic perspective has not been revealed. Concurrently, an understanding of protein-protein interactions will provide a broader perspective on the regulatory mechanism of AfLaeA in A. flagrans. This finding is critical for understanding the regulatory role of AfLaeA in the biocontrol fungus A. flagrans and establishes a foundation for developing high-efficiency nematode biocontrol agents.

Candidiasis now represents the fourth most frequent nosocomial infection both in the USA and worldwide. Candida albicans is an increasingly common threat to human health as a consequence of AIDS, steroid therapy, organ and tissue transplantation, cancer therapy, broad-spectrum antibiotics, and other immune defects. Unfortunately, these infections carry unacceptably high morbidity, mortality rates and important economic repercussions (estimated total direct cost of approximately 2 billion dollars in 1998 in US hospitals alone). This pathogen can grow both in yeast and filamentous forms and the pathogenic potential of C. albicans is intimately related to certain key processes including filamentation. Chlamydospores are considered to be a dormant form of C. albicans that remain understudied. Chlamydospores have been widely used as a diagnostic tool to separate C. albicans and C. dubliniensis from other Candida species. More recently, media have been developed that use chlamydopsore formation to separate C. albicans and C. dubliniensis from each other. Chlamydospore formation can be stimulated by hypoxic conditions but only on limited specific media types. Here, we show that anaerobic conditions are enough to drive chlamydospore formation in C. albicans on the surface of nutrient-rich agar. [ABSTRACT FROM AUTHOR]

Global banana production is affected by Fusarium wilt, a devastating disease caused by the soilborne root‐infecting fungus, Fusarium oxysporum f. sp. cubense (Foc). Fusarium wilt is notoriously difficult to manage because infection arises through complex below‐ground interactions between Foc, the plant, and the soil microbiome in the root–soil interface, defined as the rhizosphere. Interactions in the rhizosphere play a pivotal role in processes associated with pathogen development and plant health. Modulation of these processes through manipulation and management of the banana rhizosphere provides an auspicious prospect for management of Fusarium wilt. Yet, a fundamental understanding of interactions in the banana rhizosphere is still lacking. The objective of this review is to discuss the state‐of‐the‐art of the relatively scant data available on banana below‐ground interactions in relation to Fusarium wilt and, as a result, to highlight key research gaps. Specifically, we seek to understand (a) the biology of Foc and its interaction with banana; (b) the ecology of Foc, including the role of root‐exuded metabolites in rhizosphere interactions; and (c) soil management practices and how they modulate Fusarium wilt. A better understanding of molecular and ecological factors influencing banana below‐ground interactions has implications for the development of targeted interventions in the management of Fusarium wilt through manipulation of the banana rhizosphere. [ABSTRACT FROM AUTHOR]

Abstract Autophagy is an evolutionarily conserved degradation process that degrades damaged proteins to maintain homeostasis and to protect cells against stress. In this study, we identified and characterized a critical autophagy-related protein, UvAtg14, in Ustilaginoidea virens, which is the ortholog of MoAtg14 in rice blast fungus Magnaporthe oryzea. UvAtg14 is co-localized with UvAtg8 (an autophagy marker protein) and is highly expressed at 1–3 days post-inoculation. Deletion of the UvATG14 gene blocked GFP-UvAtg8 trafficking and autophagic digestion and significantly reduced mycelial growth, asexual reproduction, and virulence of U. virens. UvATG14 deletion mutants also exhibited increased sensitivity to various abiotic stresses. Our findings indicate that UvAtg14 is a key autophagic protein and contributes to mycelial growth, conidia production, and pathogenicity in U. virens.

Trichoderma strains have been successfully used in plant disease control. However, the poor stress resistance of mycelia and conidia makes processing and storage difficult. Furthermore, they cannot produce chlamydospores in large quantities during fermentation, which limits the industrialization process of chlamydospore preparation. It is important to explore an efficient liquid fermentation strategy for ensuring chlamydospore production in Trichoderma harzianum. We found that the addition of mannitol, glycine betaine, and N-acetylglucosamine (N-A-G) during liquid fermentation effectively increases the yield of chlamydospores. Furthermore, we provided evidence that chlamydospores have stronger tolerance to high temperature, ultraviolet, and hypertonic stress after the addition of mannitol and trehalose. Lipids are an important component of microbial cells and impact the stress resistance of microorganisms. We studied the internal relationship between lipid metabolism and the stress resistance of chlamydospores by detecting changes in the lipid content and gene expression. Our results showed that mannitol and trehalose cause lipid accumulation in chlamydospores and increase the unsaturated fatty acid content. In conclusion, we verified that these exogenous regulators increase the production of chlamydospores and enhance their stress resistance by regulating lipid metabolism. In addition, we believe that lipid metabolism is an important part of the chlamydospore production process and impacts the stress resistance of chlamydospores. Our findings provide clues for studying the differentiation pathway of chlamydospores in filamentous fungi and a basis for the industrial production of chlamydospores. [ABSTRACT FROM AUTHOR]

Trichoderma strains have been successfully used in plant disease control. However, the poor stress resistance of mycelia and conidia makes processing and storage difficult. Furthermore, they cannot produce chlamydospores in large quantities during fermentation, which limits the industrialization process of chlamydospore preparation. It is important to explore an efficient liquid fermentation strategy for ensuring chlamydospore production in Trichoderma harzianum. We found that the addition of mannitol, glycine betaine, and N-acetylglucosamine (N-A-G) during liquid fermentation effectively increases the yield of chlamydospores. Furthermore, we provided evidence that chlamydospores have stronger tolerance to high temperature, ultraviolet, and hypertonic stress after the addition of mannitol and trehalose. Lipids are an important component of microbial cells and impact the stress resistance of microorganisms. We studied the internal relationship between lipid metabolism and the stress resistance of chlamydospores by detecting changes in the lipid content and gene expression. Our results showed that mannitol and trehalose cause lipid accumulation in chlamydospores and increase the unsaturated fatty acid content. In conclusion, we verified that these exogenous regulators increase the production of chlamydospores and enhance their stress resistance by regulating lipid metabolism. In addition, we believe that lipid metabolism is an important part of the chlamydospore production process and impacts the stress resistance of chlamydospores. Our findings provide clues for studying the differentiation pathway of chlamydospores in filamentous fungi and a basis for the industrial production of chlamydospores.

Rice false smut caused by Villosiclava virens is a devastating disease. The smut balls contain chlamydospores, which fall onto paddy soils to become the primary inoculum. After transplanting, the chlamydospores subsequently germinate and infect rice roots. The application of a CaO‐containing fertilizer to paddy soils can inhibit the development of rice false smut disease; however, the underlying mechanism is unknown. In this study, we evaluated the suppression of chlamydospore behaviour due to CaO. Specifically, we clarified the effects of the following on chlamydospore morphology and germination: (a) pH, (b) calcium (Ca) concentration, (c) Ca concentration (pH adjusted to 6.5), and (d) CaO‐added soil solution. Germination was suppressed at pH 4 and 10, in contrast with the normal germination at pH 6, 7, and 8. Treatments with more than 10 mg/L Ca melted the outer layer of chlamydospores and suppressed germination regardless of whether the pH was adjusted to 6.5. A saturated CaO solution induced bursting of chlamydospores. Suppressed germination and a melted outer chlamydospore layer were also observed, even though the soil buffering effect initially prevented the CaO‐mediated pH increase. Furthermore, the chlamydospores within 15 mm from the CaO small lump exhibited suppressed germination in soil. In addition, there was no effect of CaO treatment on chlamydospore attachment to rice roots and hyphal invasion of rice roots in in vitro inoculation tests. These results suggest that Ca concentration is an important factor for inhibiting the occurrence of rice false smut disease. [ABSTRACT FROM AUTHOR]

Abstract Background During current submerged fermentation for microbial lipid production, the large-scale reactor operations inevitably consume substantial amounts of water and electricity for aeration, stirring, and temperature control and result in the operational costs almost exceeding the biodiesel value produced. Thus, developing a novel low-cost cultivation strategy is urgently needed by microbial lipid industry. Results The filamentous fungus Phanerochaete chrysosporium can synthesize and accumulate lipids via static solid cultivation. The conversion efficiency of substrates to lipids reaches 0.277 g/g substrate after optimization of the following cultivation factors: humidity, solid medium thickness, temperature, and rotary speed. The lipids obtained by static solid cultivation differ in component and relative content from those achieved by submerged cultivation. Laser scanning confocal microscopy reveals that numerous chlamydospores filled with lipids appear during static solid cultivation, and the fungal morphological change explains why static solid cultivation is superior in lipid yield compared with submerged fermentation. The genes coding the enzymes related to fatty acid elongation and degradation are differently expressed during static solid cultivation, which presents an answer to the appearance of abundant saturated long-chain fatty acids (93.6% in total fatty acids) in chlamydospores. In addition, engineering viability and cost–benefit analysis show that the conversion of wheat bran and glucose to lipid by the fungus is efficient. More importantly, the solid cultivation incurs only a small reactor operational cost because neither cooling water nor electrical equipment, including aerator, stirrer, and the temperature control system, is used. Conclusions This study developed a robust and cost-saving solid fermentation method without an aerator, stirrer, and temperature control system to produce microbial lipids using the chlamydospores of P. chrysosporium. Compared with conventional submerged fermentation, the solid cultivation strategy is promising because it diminishes most of the reactor operational costs, including water and power expenses.

Phanerochaete chrysosporium is a white rot fungus naturally isolated from hardwoods and widely used in environmental pollution control because it produces extracellular peroxidases. It forms chlamydospores during nitrogen starvation, which naturally occurs in the habitat of P. chrysosporium. Chlamydospores protect fungi against many stresses; the molecular basis underlying chlamydospore formation in basidiomycetes is poorly explored. Chlamydospores in P. chrysosporium have a different cell wall compared with hyphae, as confirmed by cell wall digestion and microscopy. Furthermore, this study investigated the transcriptome of P. chrysosporium in different life stages, including conidium, hypha, and chlamydospore formation, through RNA sequencing. A total of 2215 differentially expressed genes were identified during these processes. The expression patterns of genes involved in several molecular events critical for chlamydospore formation, including starch and sucrose metabolism, phosphatase and kinase, and transcription factors, were determined. This study serves as a basis for further investigating the function of chlamydospore formation in the biotechnologically relevant fungus P. chrysosporium.

Phytophthora infestans causes late blight of potato and tomato. In this study, two distinct groups of P. infestans isolates are reported based on morphological and molecular characters. The isolates were recovered from 300 single lesion leaf samples collected from potato in western and southern Ethiopia. The pathogen DNA was isolated from the single lesion dry leaves while live cultures were isolated from actively growing lesions of 100 leaves. Mitochondrial DNA (mtDNA) analysis clearly separated the isolates into two distinct groups. One of the groups corresponds to a known P. infestans mtDNA haplotype but the other is unique with a distinct Ic mtDNA haplotype and atypical morphological characters. DNA sequencing of the mtDNA confirmed that the isolates represented a previously unknown variant of P. infestans attacking Solanum tuberosum. In addition, the Ic mtDNA haplotype isolates produced papillate sporangia and chlamydospores that distinguished them from typical P. infestans isolates. All examined isolates were heterothalic and compatibility mating type A1. The novel group of the Ic mtDNA isolates constituted a single clonal population that had identical DNA fingerprint with a dominant lineage of the Ia mtDNA haplotype of P. infestans across nine microsatellite markers. This study provided new insight into a previously unknown variability within P. infestans in a region of the world where the pathogen population has rarely been studied. Further work is needed to understand the origins and significance of this finding.

The banana is one of Colombia's main export products. However, production is seriously affected by Fusarium wilt of banana, which is the most destructive disease caused by the fungus Fusarium oxysporum f. sp. cubense tropical race 4 ( Foc TR4). Currently, management strategies focus on containment and biosecurity protocols to prevent its spread to territories that are free of this disease. This study aimed to evaluate nine quaternary ammonium-based disinfectants (i.e., quaternary ammonium compounds [QACs]) in vitro in Colombia on reproductive (microconidia and macroconidia) and resistance structures (chlamydospores) of Foc race 1 (R1) and tropical race 4 (TR4), with and without soil, to determine the influence of organic matter and soil texture on the action of QACs. A method for inhibiting the action of QACs was standardized and evaluated at 1,200 ppm with a contact time of ≤30 s while evaluating the soil-inoculum and soil-disinfectant interactions. In the soil-inoculum interaction, the efficacy of QACs was 100% in the reproductive and resistance structures of Foc R1 and TR4 without soil. However, in the soil-disinfectant interaction, only QAC4 controlled the pathogen at 100%. The presence of organic matter influenced the biocidal action of the QACs, and fine textures had a greater reducing effect on the concentration. The soil decreased the efficacy of the QACs and, therefore, must be removed from contaminated boots before treatments are applied., Competing Interests: The author(s) declare no conflict of interest.

Phanerochaete chrysosporium is a white rot fungus naturally isolated from hardwoods and widely used in environmental pollution control because it produces extracellular peroxidases. It forms chlamydospores during nitrogen starvation, which naturally occurs in the habitat of P. chrysosporium. Chlamydospores protect fungi against many stresses; the molecular basis underlying chlamydospore formation in basidiomycetes is poorly explored. Chlamydospores in P. chrysosporium have a different cell wall compared with hyphae, as confirmed by cell wall digestion and microscopy. Furthermore, this study investigated the transcriptome of P. chrysosporium in different life stages, including conidium, hypha, and chlamydospore formation, through RNA sequencing. A total of 2215 differentially expressed genes were identified during these processes. The expression patterns of genes involved in several molecular events critical for chlamydospore formation, including starch and sucrose metabolism, phosphatase and kinase, and transcription factors, were determined. This study serves as a basis for further investigating the function of chlamydospore formation in the biotechnologically relevant fungus P. chrysosporium. [ABSTRACT FROM AUTHOR]

The success of Candida albicans as a pathogen relies on its ability to adapt and proliferate in different environmental niches. Pathways regulated by mitogen-activated protein kinases (MAPKs) are involved in sensing environmental conditions and developing an accurate adaptive response. Given the frequent cooperative roles of these routes in cellular functions, we have generated mutants defective in all combinations of the four described MAPKs in C. albicans and characterized its phenotype regarding sensitiveness to specific drugs, morphogenesis and interaction with host immune cells. We demonstrate that all MAPKs are dispensable in this yeast as a mutant defective in Cek1, Cek2, Mkc1 and Hog1 is viable although highly sensitive to oxidative and osmotic stress, displaying a specific pattern of sensitivity to antifungals. By comparing its phenotype with single, double and triple combinations of MAPK-deletion mutants we were able to unveil a Cek1-independent mechanism for Hog1 resistance to Congo red, and confirm the predominant effect of Hog1 on oxidative and osmotic adaptation. The quadruple mutant produces filaments under non-inducing conditions, but is unable to develop chlamydospores. Furthermore, cek1 cek2 mkc1 hog1 cells switch to the opaque state at high frequency, which is blocked by the ectopic expression of HOG1 suggesting a role of this kinase for phenotypic switching. [ABSTRACT FROM AUTHOR]

Sparassis latifolia is a valuable edible fungus cultivated in East Asia that is rich in β-glucans. Understanding the mating system and sexual life cycle is important not only for breeding programs to improve strains but also for studies on speciation and population structures. In the present study, mating experiments using monokaryons derived from two different parental strains were performed. Chi-squared test indicated satisfied Mendel segregation, which supported a tetrapolar mating system. A search in the genome for homologs to the well-defined homeodomain and pheromone/receptors, as well as frequently found flanking genes, resulted in the identification of known mating-type loci previously identified in tetrapolar basidiomycetes, each represented by two idiomorphic alleles on separate contigs. Deficiency of the β-flanking protein in S. latifolia and S. crispa around the MAT-A locus may be explained by the locus being rich in transposable elements adjacent to HD genes. Monokaryotic mycelia are characterized by a slower growth rate and a relative lack of aerial mycelia compared with the parental strain. Chlamydospores can be produced in both monokaryotic and dikaryotic mycelial stages. We provide genetic and molecular evidence for the mating system of S. latifolia , a finding that will be helpful for the cross-breeding of this mushroom. • Both genetic and molecular evidence supports the tetrapolar system of Sparassis latifolia. • β-Flanking protein is absent around the MAT-A locus in S. latifolia and Sparassis crispa. • Three transposable elements are adjacent to HD genes in S. latifolia and S. crispa. [ABSTRACT FROM AUTHOR]

Fusarium wilt of tomato caused by the ascomycetous fungus Fusarium oxysporum f. sp. lycopersici (Fol) is widespread in most tomato planting areas. Calcineurin is a heterodimeric calcium/calmodulin-dependent protein phosphatase comprised of catalytic (Cna1) and regulatory (Cnb1) subunits. Calcineurin has been studied extensively in human fungal pathogens, but less is known about its roles in plant fungal pathogens. It is known that calcineurin regulates fungal calcium signaling, growth, drug tolerance, and virulence. However, the roles of calcineurin in Fol have not yet been characterized. In this study, we deleted calcineurin CNA1 and CNB1 genes to characterize their roles in conidiation, chlamydospore formation and virulence in Fol. Our results revealed that both cna1 and cnb1 mutants show defects in calcineurin phosphatase activity, vegetative growth and conidiation as compared to the wild type. Furthermore, calcineurin mutants exhibited blunted and swollen hyphae as observed by scanning electron microscopy. Interestingly, we found that Fol calcineurin is critical for chlamydospore formation, a function of calcineurin previously undocumented in the fungal kingdom. According to transcriptome analysis, the expression of 323 and 414 genes was up- and down-regulated, respectively, in both cna1 and cnb1 mutants. Based on the pathogen infection assay, tomato plants inoculated with cna1 or cnb1 mutant have a dramatic reduction in disease severity, indicating that calcineurin has a vital role in Fol virulence. In conclusion, our findings suggest that Fol calcineurin is required, at least in part, for phosphatase activity, vegetative growth, conidiation, chlamydospore formation, and virulence.

Fusarium wilt of tomato caused by the ascomycetous fungus Fusarium oxysporum f. sp. lycopersici (Fol) is widespread in most tomato planting areas. Calcineurin is a heterodimeric calcium/calmodulin-dependent protein phosphatase comprised of catalytic (Cna1) and regulatory (Cnb1) subunits. Calcineurin has been studied extensively in human fungal pathogens, but less is known about its roles in plant fungal pathogens. It is known that calcineurin regulates fungal calcium signaling, growth, drug tolerance, and virulence. However, the roles of calcineurin in Fol have not yet been characterized. In this study, we deleted calcineurin CNA1 and CNB1 genes to characterize their roles in conidiation, chlamydospore formation and virulence in Fol. Our results revealed that both cna1 and cnb1 mutants show defects in calcineurin phosphatase activity, vegetative growth and conidiation as compared to the wild type. Furthermore, calcineurin mutants exhibited blunted and swollen hyphae as observed by scanning electron microscopy. Interestingly, we found that Fol calcineurin is critical for chlamydospore formation, a function of calcineurin previously undocumented in the fungal kingdom. According to transcriptome analysis, the expression of 323 and 414 genes was up- and down-regulated, respectively, in both cna1 and cnb1 mutants. Based on the pathogen infection assay, tomato plants inoculated with cna1 or cnb1 mutant have a dramatic reduction in disease severity, indicating that calcineurin has a vital role in Fol virulence. In conclusion, our findings suggest that Fol calcineurin is required, at least in part, for phosphatase activity, vegetative growth, conidiation, chlamydospore formation, and virulence. [ABSTRACT FROM AUTHOR]

Species of Endogonaceae (Endogonales, Mucoromycotina) are characterized by the formation of relatively large sporocarps and zygosporangia. Numerous species in this family remain undescribed or have unclear phylogenetic positions. In Asia specifically, the species diversity of this family is almost completely unknown. However, many mycobionts of bryophytes belonging to several novel clades in Endogonaceae have recently been identified phylogenetically. Therefore, establishing a robust taxonomic system for this family is essential. We obtained numerous sporocarps of undescribed Endogonaceae-like species from the Japanese islands. Morphological observation and multilocus phylogenetic analysis of nuc 18S rDNA (18S), nuc 28S rDNA (28S), and portions of two nuclear protein-coding regions—translation elongation factor 1-alpha (tef1) and RNA polymerase II large subunit (rpb1)—from these species resulted in the description of one new species each of Endogone and Jimgerdemannia and two new species of Vinositunica, gen. nov. Because Vinositunica is characterized by purplish sporocarps and red-wine-colored chlamydospores up to 700 μm in diameter, we emended the definition of Endogonaceae. [ABSTRACT FROM AUTHOR]

In order to develop a simple method for crossing two transformants, we first attempted to elucidate the fusion type (self-compatibility or -incompatible) of Phanerochaete sordida YK-624. Two transformants expressing green or red fluorescent protein derived from an auxotrophic mutant were constructed. Each recombinant protein fluoresced by expression as a fused protein with glyceraldehyde-3-phosphate dehydrogenase. On co-culture of both transformants, a number of sequential hyphal cells emitting dual fluorescence were formed at the contact areas of both hyphae. Some of the single cells isolated as protoplasts and chlamydospore from the co-cultures also expressed these fluorescent proteins. These results suggest that P. sordida YK-624 possesses a self-compatible fusion system. In addition, transformant strains with different fluorescence derived from this fungus can readily undergo self-fusion and nuclear interchange events by confrontational and mixed cultivation, and we developed a simple method that allows fused cells to be isolated as chlamydospores. [ABSTRACT FROM AUTHOR]

Bioversity of fungal antagonist, Trichoderma from different locations of Nilgiri district of Tamil Nadu, India, were characterized through molecular methods. Thirty four isolates were tentatively identified as Trichoderma and confirmed upto species level through molecular tools. PCR amplification of the 18s-28s rRNA gene region revealed that all thirty four isolates produced amplicon size of 600bp and were further confirmed through sequencing. The sequences of various Trichoderma spp. were compared with both NCBI and TrichOKEY database to validate their molecular identity. Among 34 isolates, 31 isolates were identified as T. asperellum (KT462693, KU361372, KX533978 to KX533999, KX523262 to KX523264, KX555650, KX147092 to KX147094, KX5334000), 2 isolates as T. harzianum (KX533989, KX533990) and 1 isolate as T. virens (KU666466) through NCBI data base. However, all the T. asperellum isolates identified through NCBI database were identified as T. koningiopsis using TricHOKEY data base. Identity of T. harzianum isolates (TRI 35 and TRI 36) and T. virens isolate (TRI 37) were same in both NCBI and TricHOKEY database. Antagonistic assay with diverse species of Trichoderma revealed that T. virens (TRI 37) was effective in inhibiting the radial growth of Pythium aphanidermatum (87.78%) followed by T. harzianum (TRI 35), (TRI 36) and T. asperellum (TRI 9) in vitro. The effective isolates T. virens (TRI 37), T. harzianum (TRI 35, TRI 36) and T. asperellum (TRI 9) were compatible with each other. Biopriming of cucumber seeds with talc based formulation of the consortia comprising of T. virens isolate ( TRI 37), T. harzianum isolates (TRI 35 and TRI 36) and T. asperellum TRI 9 @ 108 cfu/g and soil application suppressed damping off to an extent of 76.82% over untreated control.

Rice false smut fungus (teleomorph: Villosiclava virens; anamorph: Ustilaginoidea virens) can generate chlamydospores and survive winter under field conditions. The chlamydospore is considered as an important infection source of the disease. However, little is known about the regulatory mechanism of the chlamydospore production. In this study, we identified a defective homeobox transcription factor (designated as UvHOX2) gene in a U. virens random insertional mutant B-766 that could not form chlamydospores. To confirm the regulatory function of UvHOX2, an Agrobacterium tumefaciens mediated transformation- and CRISPR/Cas9- based targeted gene replacement method was developed. The UvHox2 deletion mutants completely failed to produce chlamydospores, showed reduced conidia production and decreased virulence, and was hyper-sensitive to oxidative, osmotic, and cell wall stresses. We confirmed that UvHOX2 is located in the nuclei of U. virens, and the expression of UvHox2 was the strongest during the early stage of chlamydospore and conidium formation. Global transcription pattern of UvHOX2 was also determined by RNA-seq in this study, and several genes that might be down-stream of UvHOX2 regulation were identified. The results will better our understanding of the molecular mechanism of chlamydospore formation in U. virens as a model fungus.

Rice false smut fungus (teleomorph: Villosiclava virens ; anamorph: Ustilaginoidea virens) can generate chlamydospores and survive winter under field conditions. The chlamydospore is considered as an important infection source of the disease. However, little is known about the regulatory mechanism of the chlamydospore production. In this study, we identified a defective homeobox transcription factor (designated as UvHOX2) gene in a U. virens random insertional mutant B-766 that could not form chlamydospores. To confirm the regulatory function of UvHOX2, an Agrobacterium tumefaciens mediated transformation- and CRISPR/Cas9- based targeted gene replacement method was developed. The UvHox2 deletion mutants completely failed to produce chlamydospores, showed reduced conidia production and decreased virulence, and was hyper-sensitive to oxidative, osmotic, and cell wall stresses. We confirmed that UvHOX2 is located in the nuclei of U. virens , and the expression of UvHox2 was the strongest during the early stage of chlamydospore and conidium formation. Global transcription pattern of UvHOX2 was also determined by RNA-seq in this study, and several genes that might be down-stream of UvHOX2 regulation were identified. The results will better our understanding of the molecular mechanism of chlamydospore formation in U. virens as a model fungus. [ABSTRACT FROM AUTHOR]

Context: Candida, leukoplakia and oral squamous cell carcinoma. Aims: The aim of the present study has assessed the association of Candida albicans with normal control group, leukoplakia and oral squamous cell carcinoma lesions of the oral cavity by using cornmeal broth +5% milk and milk serum liquid culture media. Settings and Design: Thepresent study comprised of total 80 patients,which included, 30 patients of Leukoplakia, 30 patients of oral squamous cell carcinoma and normal control group comprised of 20 healthy individuals who were not having any relevant medical, dental and habit history. Subjects and Methods: Saliva and Biopsy was taken from clinically suspected leukoplakia and oral squamous cell carcinoma lesions for the confirmation of histopathological diagnosis. Saliva samples were inoculated for fungal growth in Sabouraud Dextrose Agar, and culture-positive samples had undergone for the germ tube test. Germ tube-positive samples were further taken for chlamydospore production in milk serum and cornmeal broth +5% milk media separately at 8 and 16 h. Statistical Analysis Used: Chi-square test, Fischer extract test. Results: In the normal control group, no fungus growth was found; however, leukoplakia and oral squamous cell carcinoma showed fungus growth with positive germ tube test and chlamydospore formation; the result also showed rapid and quantitatively more chlamydospore formation in cornmeal broth +5% milk in comparison to serum milk culture media. There was no growth of Candida in mild and moderate form of leukoplakia. Whereas in a severe form of leukoplakia, there was more quantitative chlamydospore growth in cornmeal broth +5% milk liquid media. Conclusions: In this study, we have tried to compare the efficacy of cornmeal broth +5% milk and milk serum for the identification of C. albicans. Both the culture media were able to promote the growth of chlamydospore in C. albicans. Among different grades of leukoplakia, the growth of C. albicans was seen in severe dysplastic patient only while mild and moderate dysplasia showed no Candidal growth. [ABSTRACT FROM AUTHOR]

The success of Candida albicans as a pathogen relies on its ability to adapt and proliferate in different environmental niches. Pathways regulated by mitogen-activated protein kinases (MAPKs) are involved in sensing environmental conditions and developing an accurate adaptive response. Given the frequent cooperative roles of these routes in cellular functions, we have generated mutants defective in all combinations of the four described MAPKs in C. albicans and characterized its phenotype regarding sensitiveness to specific drugs, morphogenesis and interaction with host immune cells. We demonstrate that all MAPKs are dispensable in this yeast as a mutant defective in Cek1, Cek2, Mkc1 and Hog1 is viable although highly sensitive to oxidative and osmotic stress, displaying a specific pattern of sensitivity to antifungals. By comparing its phenotype with single, double and triple combinations of MAPK-deletion mutants we were able to unveil a Cek1-independent mechanism for Hog1 resistance to Congo red, and confirm the predominant effect of Hog1 on oxidative and osmotic adaptation. The quadruple mutant produces filaments under non-inducing conditions, but is unable to develop chlamydospores. Furthermore, cek1 cek2 mkc1 hog1 cells switch to the opaque state at high frequency, which is blocked by the ectopic expression of HOG1 suggesting a role of this kinase for phenotypic switching.

Arthrobotrys flagrans ( Duddingtonia flagrans ) is a typical nematode-trapping fungus which has been used for nematode biocontrol. The global regulator LaeA is widely distributed in filamentous fungi and plays a crucial role in secondary metabolism and development in addition to pathogenicity in fungal pathogens. In this study, the chromosome-level genome of A. flagrans CBS 565.50 was sequenced and homologous sequences of LaeA were identified in A. flagrans . A. flagrans LaeA ( AfLaeA ) knockout resulted in slower hyphal growth and a smoother hyphal surface. Importantly, deletion of AfLaeA resulted in the absence of chlamydospores and attenuated glycogen and lipid accumulation in hyphae. Similarly, disruption of the AfLaeA gene led to fewer traps and electron-dense bodies, lower protease activity, and a delay in capturing nematodes. The AfLaeA gene had a large effect on the secondary metabolism of A. flagrans , and both the deletion and overexpression of AfLaeA could yield new compounds, whereas some compounds were lost due to the absence of the AfLaeA . Protein-protein interactions between AfLaeA and another eight proteins were detected. Furthermore, transcriptome data analysis showed that 17.77% and 35.51% of the genes were influenced by the AfLaeA gene on days 3 and 7, respectively. AfLaeA gene deletion resulted in the higher expression level of the artA gene cluster, and multiple differentially expressed genes involved in glycogen and lipid synthesis and metabolism showed opposite expression patterns in wild-type and Δ AfLaeA strains. In summary, our results provide novel insights into the functions of AfLaeA in mycelial growth, chlamydospore production, pathogenicity, secondary metabolism, and energy metabolism in A. flagrans . IMPORTANCE The regulation of biological functions, such as the secondary metabolism, development, and pathogenicity of LaeA, has been reported in multiple fungi. But to date, no study on LaeA in nematode-trapping fungi has been reported. Moreover, it has not been investigated whether or not LaeA is involved in energy metabolism and chlamydospore formation has not been investigated. Especially in the formation mechanism of chlamydospores, several transcription factors and signaling pathways are involved in the production of chlamydospores, but the mechanism of chlamydospore formation from an epigenetic perspective has not been revealed. Concurrently, an understanding of protein-protein interactions will provide a broader perspective on the regulatory mechanism of AfLaeA in A. flagrans . This finding is critical for understanding the regulatory role of AfLaeA in the biocontrol fungus A. flagrans and establishes a foundation for developing high-efficiency nematode biocontrol agents., Competing Interests: The authors declare no conflict of interest.

Various types of other fungi colonize glomeromycotinan (Mucoromycota) spores in the Early Devonian Rhynie chert. However, relatively few of these associations have been described and evaluated in detail. One particular type of glomoid spore located in degrading land plant axes from the Rhynie chert provides evidence of (simultaneous) interaction with three different fungi. Massive callosities occur around the intrusion filaments of a chytrid-like parasite with epibiotic sporangia, while the hyphae of a delicate mycelial fungus extend into the spore lumen without triggering a recognizable host response. Several spores show large numbers of inwardly directed projections, which are regularly distributed and consist of a short hyphal branch encased in host wall material. The projections represent the penetration sites of a distinctive, mycelial fungus that forms a mantle-like hyphal sheath around the spores. This type of fungal interaction with glomeromycotinan spores has not previously been reported, and thus expands our knowledge of the numerous interfungal relationships that existed in early continental ecosystems. [ABSTRACT FROM AUTHOR]

Bioversity of fungal antagonist, Trichoderma from different locations of Nilgiri district of Tamil Nadu, India, were characterized through molecular methods. Thirty four isolates were tentatively identified as Trichoderma and confirmed upto species level through molecular tools. PCR amplification of the 18s-28s rRNA gene region revealed that all thirty four isolates produced amplicon size of 600bp and were further confirmed through sequencing. The sequences of various Trichoderma spp. were compared with both NCBI and TrichOKEY database to validate their molecular identity. Among 34 isolates, 31 isolates were identified as T. asperellum (KT462693, KU361372, KX533978 to KX533999, KX523262 to KX523264, KX555650, KX147092 to KX147094, KX5334000), 2 isolates as T. harzianum (KX533989, KX533990) and 1 isolate as T. virens (KU666466) through NCBI data base. However, all the T. asperellum isolates identified through NCBI database were identified as T. koningiopsis using TricHOKEY data base. Identity of T. harzianum isolates (TRI 35 and TRI 36) and T. virens isolate (TRI 37) were same in both NCBI and TricHOKEY database. Antagonistic assay with diverse species of Trichoderma revealed that T. virens (TRI 37) was effective in inhibiting the radial growth of Pythium aphanidermatum (87.78%) followed by T. harzianum (TRI 35), (TRI 36) and T. asperellum (TRI 9) in vitro. The effective isolates T. virens (TRI 37), T. harzianum (TRI 35, TRI 36) and T. asperellum (TRI 9) were compatible with each other. Biopriming of cucumber seeds with talc based formulation of the consortia comprising of T. virens isolate ( TRI 37), T. harzianum isolates (TRI 35 and TRI 36) and T. asperellum TRI 9 @ 108 cfu/g and soil application suppressed damping off to an extent of 76.82% over untreated control. [ABSTRACT FROM AUTHOR]