Screening Proteins That Interact With AcHog1 and the Functional Analysis of AcSko1 in Aspergillus cristatus.

Aspergillus cristatus is a dominant fungus formed during the "flowering" process of Fuzhuan brick tea. Previous research has established that the sporulation of Aspergillus nidulans, a model organism of filamentous fungi, is regulated by light. However, the sporulation of A. cristatus is dependent on osmotic stress. In a previous study, we used pull-down and mass spectrometry to identify proteins that interacted with AcHog1 in A. cristatus when cultured under different conditions of osmotic stress. In the present study, we analyzed the proteins we identified previously to investigate their functional role. The AA1E3BER4 protein was located downstream of Hog1 in the HOG branch pathway and was identified that was regulated by AcHog1. Furthermore, yeast two-hybrid analysis showed that AA1E3BER4 interacted with AcHog1. In addition, we knocked out and complemented the Acsko1 gene encoding the AA1E3BER4 protein. We found that the number of sexual and asexual spores were downregulated by 3.81- and 4.57-fold, respectively, in the ΔAcsko1 strain. The sensitivity of the ΔAcsko1 strain to sorbitol and sucrose, as regulators of osmotic stress, increased, and the sensitivity to high sucrose was higher than that of sorbitol. Acsko1 also regulated the response of A. cristatus to oxidative stress, Congo red, and SDS (sodium dodecyl sulfate). In addition, the deletion of Acsko1 significantly increased the pigment of the ΔAcsko1 strain. This is the first study to report the role of the sko1 gene in oxidative stress, stress-induced damage to the cell wall, and pigment in Aspergillus cristatus.

Leaf spot on Photinia × fraseri caused by Neopestalotiopsis asiatica in China.

Photinia × fraseri is a well-known evergreeen ornamental tree. Owing to its flower-like red leaves and its ability to tolerate stressful environments, P. fraseri is widely cultured as a fast-growing hedge in southern China. From July to September in 2021, a disease with symptoms similar to leaf spot was extensively observed on P. fraseri in Daozhen county (28° 51 'N, 107° 57 'E), Zunyi, Guizhou province, China. About 500 plants were surveyed and the incidence of leaf spot on P. fraseri leaves was 35% to 70%, significantly reducing the ornamental and economic value. The symptomatic leaves displayed irregular, watery dark brown lesions with black conidiomata in gray centers, and 10 symptomatic leaves were collected from 10 trees. After surface sterilization (0.5 min in 75% ethanol and 2 min in 3% NaOCl, washed three times with sterilized distilled water) (Fang 2007), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on potato dextrose agar (PDA) and incubated at 25°C for about 7 days. Three single-spore isolates, GZAAS 21-0327, GZAAS 21-0328 and GZAAS 21-0329, were obtained, which were identical in morphology and molecular analysis. Therefore, the representative isolate GZAAS 21-0328 was used for further study. The pathogenicity of GZAAS 21-0328 was tested through a pot assay. Ten healthy plants were scratched with a sterilized needle on the leaves. Plants were inoculated by spraying a spore suspension (106 spores mL-1) of GZAAS 21-0328 onto leaves until runoff, and the control leaves sprayed with sterile water. The plants were maintained at 28°C with high relative humidity (95%) in a growth chamber. The pathogenicity test was carried out three times (Fang 2007). The symptoms developed on all inoculated leaves but not on the control leaves. The lesions were first visible 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after 15 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses (ITS, TUB and TEF) from the infected leaves but not from the noninoculated leaves. Results of pathogenicity experiments of isolated fungi fulfilled Koch's postulates. Fungal colonies on PDA were villiform, creamy-white and sparse aerial mycelium on the surface with black, gregarious conidiomata. The conidia were fusoid, ellipsoid, straight to slightly curved, 4-septate, septa darker than the rest of the cell, and 23.0 (21.0 to 27.0) × 6.0 (5.0 to 7.0) µm (n=50). The morphological features were consistent with the descriptions of Neopestalotiopsis asiatica Maharachch. & K.D. Hyde (Maharachchikumbura et al. 2012; Maharachchikumbura et al. 2014; Farr et al. 2022). The pathogen was confirmed to be N. asiatica by amplification and sequencing of the internal transcribed spacer region (ITS), the partial ß-tubulin (TUB) and partial translation elongation factor 1-alpha (TEF) genes using primers ITS4/ITS5, T1/Bt-2b and EF1-728F/EF-2, respectively (Maharachchikumbura et al. 2014). The sequences of PCR products were deposited in GenBank with accession numbers OK563071 (ITS), OK584020 (TUB) and OK663023 (TEF). BLAST searches of the obtained sequences revealed 100% (482/482 nucleotides), 99.05% (419/421 nucleotides), and 99.33% (891/897 nucleotides) homology with those of N. asiatica in GenBank (JX398983, JX399018 and JX399049, respectively). Phylogenetic analysis (MEGA 6.0) using the maximum likelihood method placed the isolate GZAAS 21-0328 in a well-supported cluster with N. asiatica. The pathogen was thus identified as N. asiatica based on the morphological characterization and molecular analyses. To our knowledge, this is the first report of leaf spot on P. fraseri caused by N. asiatica in China. This study provides valuable information for the identification and control of the leaf spot on Photinia × fraseri.

The role of Acpbs2 in the asexual sporulation, stress response and carbon metabolism of Aspergillus cristatus.

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea, hypotonic conditions only induced its sexual development to produce ascospores, while hypertonic conditions only induced its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the roles of Acpbs2, which is homologous to pbs2 from Saccharomyces cerevisiae, in sporulation, stress responses, the color of colonies, and carbon metabolism were explored in A. cristatus. Deletion mutants of Acpbs2 were obtained by homologous recombination. The time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in hypertonic media in ΔAcpbs2 by phenotypic observations, indicating that Acpbs2 plays a positive role in asexual development. Stress sensitivity tests showed that the order of the sensitivity of ΔAcpbs2 to different osmotic regulators was 3 M NaCl > 3 M sucrose > 3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. The growth of the Acpbs2 deletion mutant was inhibited under alkaline-pH stress, indicating that Acpbs2 is involved in high pH stress tolerance. Additionally, compared with the wild type, the colony color of the Acpbs2 deletion mutant became lighter. All the above developmental defects were reversed by the reintroduction of the Acpbs2 gene in ΔAcpbs2. Transcriptome data showed that Acpbs2 regulated the expression of several genes related to conidial development, osmotic stress, oxidative stress, and carbon metabolism. More importantly, the interaction between Acpbs2 and its downstream gene Achog1 was verified by yeast two-hybrid assays. We speculated that this interaction might regulate the osmotic stress response, the oxidative stress response, and asexual sporulation in A. cristatus, which will be one of the focuses of our future research.

First Report of Colletotrichum boninense Causing Anthracnose on Rosa chinensis in China.

Chinese rose (Rosa chinensis Jacq.) is cultivated for edible flowers in southwestern China (Zhang et al. 2014). In March 2020, a leaf spot disease was observed on about 3-5% leaves of Chinese rose cultivar 'Mohong' in Guizhou Botanical Garden (26°37' 45'' N, 106°43' 10'' E), Guiyang, Guizhou province, China. The symptomatic plants displayed circular, dark brown lesions with black conidiomata in grey centers on leaves, and leaf samples were collected. After surface sterilization (0.5 min in 75% ethanol and 2 min in 3% NaOCl, washed 3 times with sterilized distilled water) (Fang 2007), small pieces of symptomatic leaf tissue (0.3 × 0.3 cm) were plated on potato dextrose agar (PDA) and incubated at 28oC for about 7 days. Two single-spore isolates, GZUMH01 and GZUMH02, were obtained, which were identical in morphology and molecular analysis. Therefore, the representative isolate GZUMH01 was used for further study. The pathogenicity of GZUMH01 was tested through a pot assay. Ten healthy plants were scratched with a sterilized needle on the leaves. Plants were inoculated by spraying a spore suspension (106 spores ml-1) onto leaves until runoff, and the control leaves sprayed with sterile water. The plants were maintained at 25°C with high relative humidity (90 to 95%) in a growth chamber. The pathogenicity test was carried out three times using the method described in Fang (2007). The symptoms developed on all inoculated leaves but not on the control leaves. The lesions were first visible 48 h after inoculation, and typical lesions similar to those observed on field plants after 7 days. The same fungus was re-isolated from the infected leaves but not from the non-inoculated leaves, fulfilling Koch's postulates. Fungal colonies on PDA were villiform and greyish. The conidia were abundant, oval-ellipsoid, aseptate, 15.8 (13.7 to 18.8) × 5.7 (4.3 to 6.8) µm. The fungal colonies, hyphae, and conidia were consistent with the descriptions of Colletotrichum boninense Moriwaki, Toy. Sato & Tsukib. (Damm et al. 2012; Moriwaki et al. 2003). The pathogen was confirmed to be C. boninense by amplification and sequencing of the internal transcribed spacer region (ITS), the glyceraldehyde-3-phosphate dehydrogenase (GADPH), actin (ACT), and chitin synthase 1 (CHS-1) genes using primers ITS1/ITS4, GDF1/GDR1, ACT512F/ACT783R, and CHS-79F/CHS-345R, respectively (Damm et al. 2012; Moriwaki et al. 2003). The sequences of the PCR products were deposited in GenBank with accession numbers MT845879 (ITS), MT861006 (GADPH), MT861007 (ACT), and MT861008 (CHS-1). BLAST searches of the obtained sequences of the ITS, GADPH, ACT, and CHS-1 genes revealed 100% (554/554 nucleotides), 100% (245/245 nucleotides), 97.43% (265/272 nucleotides), and 99.64% (279/280 nucleotides) homology with those of C. boninense in GenBank (JQ005160, JQ005247, JQ005508, and JQ005334, respectively). Phylogenetic analysis (MEGA 6.0) using the maximum likelihood method placed the isolate GZUMH01 in a well-supported cluster with C. boninense. The pathogen was thus identified as C. boninense based on its morphological and molecular characteristics. To our knowledge, this is the first report of the anthracnose disease on R. chinensis caused by C. boninense in the world.

Characterizing the role of the zinc finger transcription factor AcrpnR in governing development in Aspergillus cristatus.

Filamentous fungi reproduce sexually or asexually, and the developmental processes are strictly regulated by a variety of transcription factors. In this study, we characterized a zinc finger transcription factor, called AcrpnR, in Aspergillus cristatus (GME2916). The ∆AcrpnR strain exhibited decreased asexual reproduction and increased cleistothecium production. The complementation strain showed restoration of these phenotypic differences. Overexpression of AcrpnR resulted in enhanced asexual development and delayed and inhibited sexual reproduction, suggesting that AcrpnR is required for proper asexual and sexual development in A. cristatus. In addition, AcrpnR positively regulated the expression of genes of the central regulatory pathway of conidiation and negatively regulated the expression of sex-related genes. Overall, these results demonstrate that AcrpnR is essential for maintaining a balance between asexual and sexual development.

A Comparison of Isolation Methods for Black Fungi Degrading Aromatic Toxins.

The prevalence of black fungi in the order Chaetothyriales has often been underestimated due to the difficulty of their isolation. In this study, three methods which are often used to isolate black fungi are compared. Enrichment on aromatic hydrocarbon appears effective in inhibiting growth of cosmopolitan microbial species and allows appearance of black fungi. We miniaturized the method for high-throughput purposes. The new procedure saves time, consumes less space and can process multiple samples simultaneously.

Comparative transcriptome analysis of the calcium signaling and expression analysis of sodium/calcium exchanger in Aspergillus cristatus.

Aspergillus cristatus develops into various stages under different Na concentrations: the sexual stage in 0.5 M NaCl and asexual development stage in 3 M NaCl. In order to explore whether the Ca2+ signaling pathway in A. cristatus responded to the changes in the salt stress, we analyzed the gene expression levels in A. cristatus respectively cultured in 0.5 M NaCl and 3 M NaCl. According to the BLAST analysis results, we identified 25 Ca2+ -signaling proteins in A. cristatus. The expression levels of most genes involved in the Ca2+ -signaling pathway in A. cristatus cultured in different salt concentrations showed significant differences, indicating that the Ca2+ signaling pathway was involved in the response to the changes in the salt stress. In yeasts, only calcium ion influx proteins were reported to be involved in the response to the changes in the salt stress. So far, the protein for the exchanger of calcium/sodium ions has not been reported. Therefore, we obtained the sodium/calcium exchanger (termed NCX) proteins from the KEGG Database. The ncx gene of A. cristatus was cloned and characterized. The full length of ncx gene is 3055 bp, including a 2994-bp open reading frame encoding 994 amino acids. The expression levels of ncx in the sexual development stage and asexual development stage were respectively ∼8.94 times and ∼2.57 times of that in the hyphal formation stage. Therefore, we suggested that ncx gene was up-regulated to resist the sodium stress. The study results provide the basis for further exploring the Ca2+ -signaling mechanism and ion exchanger mechanism.

Comparative Transcriptome Sequence Analysis of Sporulation-Related Genes of Aspergillus cristatus in Response to Low and High Osmolarity.

Aspergillus cristatus undergoes sexual and asexual development under conditions of low and high osmotic pressure, respectively. In this study, the expression levels of 107 genes associated with sexual and asexual development were analysed under conditions of low and high osmotic pressure by RNA sequencing. The results showed that 37 genes were up-regulated and other genes were down-regulated under conditions of high osmotic pressure, with most of the up-regulated genes associated with asexual development and most down-regulated genes associated with sexual development. These results suggest that osmotic pressure regulated sexual and asexual development of A. cristatus by controlling the expression levels of key genes. Meanwhile, there were differences in the expression levels of key genes associated with the regulation of sexual and asexual development between A. cristatus and Aspergillus nidulans. Moreover, we verified the reliability of the results by quantitative real-time polymerase chain reaction analysis of some key genes. In this study, the relationship between sporulation-related genes and osmotic pressure at the transcriptome level were analysed, which indicated that A. cristatus was a useful model organism for the study of osmotic pressure regulation on sexual and asexual development.

Comparative genomic and transcriptomic analyses of the Fuzhuan brick tea-fermentation fungus Aspergillus cristatus.

BACKGROUND: Aspergillus cristatus is the dominant fungus involved in the fermentation of Chinese Fuzhuan brick tea. Aspergillus cristatus is a homothallic fungus that undergoes a sexual stage without asexual conidiation when cultured in hypotonic medium. The asexual stage is induced by a high salt concentration, which completely inhibits sexual development. The taxon is therefore appropriate for investigating the mechanisms of asexual and sexual reproduction in fungi. In this study, de novo genome sequencing and analysis of transcriptomes during culture under high- and low-osmolarity conditions were performed. These analyses facilitated investigation of the evolution of mating-type genes, which determine the mode of sexual reproduction, in A. cristatus, the response of the high-osmolarity glycerol (HOG) pathway to osmotic stimulation, and the detection of mycotoxins and evaluation of the relationship with the location of the encoding genes. RESULTS: The A. cristatus genome comprised 27.9 Mb and included 68 scaffolds, from which 10,136 protein-coding gene models were predicted. A phylogenetic analysis suggested a considerable phylogenetic distance between A. cristatus and A. nidulans. Comparison of the mating-type gene loci among Aspergillus species indicated that the mode in A. cristatus differs from those in other Aspergillus species. The components of the HOG pathway were conserved in the genome of A. cristatus. Differential gene expression analysis in A. cristatus using RNA-Seq demonstrated that the expression of most genes in the HOG pathway was unaffected by osmotic pressure. No gene clusters associated with the production of carcinogens were detected. CONCLUSIONS: A model of the mating-type locus in A. cristatus is reported for the first time. Aspergillus cristatus has evolved various mechanisms to cope with high osmotic stress. As a fungus associated with Fuzhuan tea, it is considered to be safe under low- and high-osmolarity conditions.

The growth and medicinal quality of Epimedium wushanense are improved by an isolate of dark septate fungus.

CONTEXT: Seven dark-septate endophytic (DSE) fungi have been isolated from the roots of Epimedium wushanense T. S. Ying (Berberidaceae), an important medicinal plant with various pharmacological activities. OBJECTIVE: The current study explores the effects of seven DSE fungi on the growth and accumulation of bioactive compounds in E. wushanense. MATERIALS AND METHODS: Each 1-month-old E. wushanense seedling was inoculated with one of the seven DSE fungi and was grown under greenhouse conditions for 90 d. The molecular identification of the fungi was based on the ITS1-5.8S-ITS2 nuclear ribosomal gene cluster. RESULTS: The results showed that the influence of DSE fungi inoculation varied between strains. Inoculation with DSE8 not only significantly enhanced plant height, root length, leaf area, leaf number, and shoot and root biomass but also improved the total flavonoid and icariin content, with an increase ranging from 20.24% to 237.97%. Three of the seven DSE fungi caused the inoculated plants to die, and the remaining three DSE strains showed neutral or negative effects on plant growth and the accumulation of bioactive compounds. According to the ITS sequence, DSE8 is congeneric to the genus Leptodontidium. DISCUSSION AND CONCLUSION: The findings indicate that application of DSE8 may be valuable to facilitate the cultivation of E. wushanense with a higher biomass and improved medicinal quality.

Acptp2,3 participates in the regulation of spore production, stress response, and pigments synthesis in Aspergillus cirstatus.

Background: Aspergillus cristatus was a filamentous fungus that produced sexual spores under hypotonic stress and asexual spores under hypertonic stress. It could be useful for understanding filamentous fungi's sporulation mechanism. Previously, we conducted functional studies on Achog1, which regulated the hyperosmotic glycerol signaling (HOG) pathway and found that SI65_02513 was significantly downregulated in the transcriptomics data of ΔAchog1 knockout strain. This gene was located at multiple locations in the HOG pathway, indicating that it might play an important role in the HOG pathway of A. cristatus. Furthermore, the function of this gene had not been identified in Aspergillus fungi, necessitating further investigation. This gene's conserved domain study revealed that it has the same protein tyrosine phosphatases (PTPs) functional domain as Saccharomyces cerevisiae, hence SI65_02513 was named Acptp2,3. Methods: The function of this gene was mostly validated using gene knockout and gene complementation approaches. Knockout strains exhibited sexual and asexual development, as well as pigments synthesis. Morphological observations of the knockout strain were carried out under several stress conditions (osmotic stress, oxidative stress, Congo Red, and sodium dodecyl sulfate (SDS). Real-time fluorescence polymerase chain reaction (PCR) identified the expression of genes involved in sporulation, stress response, and pigments synthesis. Results: The deletion of Acptp2,3 reduced sexual and asexual spore production by 4.4 and 4.6 times, demonstrating that Acptp2,3 positively regulated the sporulation of A. cristatus. The sensitivity tests to osmotic stress revealed that ΔAcptp2,3 strains did not respond to sorbitol-induced osmotic stress. However, ΔAcptp2.3 strains grew considerably slower than the wild type in high concentration sucrose medium. The ΔAcptp2,3 strains grew slower than the wild type on media containing hydrogen peroxide, Congo red, and SDS. These findings showed that Acptp2,3 favorably controlled osmotic stress, oxidative stress, and cell wall-damaging chemical stress in A. cristatus. Deleting Acptp2,3 resulted in a deeper colony color, demonstrating that Apctp2,3 regulated pigment synthesis in A. cistatus. The expression levels of numerous stress-and pigments-related genes matched the phenotypic data. Conclusion: According to our findings, Acptp2,3 played an important role in the regulation of sporulation, stress response, and pigments synthesis in A. cristatus. This was the first study on the function of PTPs in Aspergillus fungi.

Phylogeny and taxonomy of two new species in Dictyosporiaceae (Pleosporales, Dothideomycetes) from Guizhou, China.

Two novel species within the family Dictyosporiaceae are described and illustrated from terrestrial habitats on dead culms of bamboo and an unidentified plant, respectively. Through morphological comparisons and the multi-locus phylogenetic analyses of combined LSU, ITS, SSU, and tef1-α sequence dataset, two species, Gregaritheciumbambusicola, Pseudocoleophomaparaphysoidea are identified. Phylogenetically, both species clustered into a monophyletic clade with strong bootstrap support. Gregaritheciumbambusicola sp. nov. can be distinguished from other species within the genus based on its almost straight ascospores. Pseudocoleophomaparaphysoidea sp. nov. differs from other species in its conidiogenous cells intermixed with paraphyses, longer conidiogenous cells and larger conidia. The identification of this lineage contributes to our understanding of the classification of Dictyosporiaceae.

A comparative proteomic analysis of Pinellia ternata leaves exposed to heat stress.

Pinellia ternata is an important traditional Chinese medicinal plant. The growth of P. ternata is sensitive to high temperatures. To gain a better understanding of heat stress responses in P. ternata, we performed a comparative proteomic analysis. P. ternata seedlings were subjected to a temperature of 38 °C and samples were collected 24 h after treatment. Increased relative ion leakage and lipid peroxidation suggested that oxidative stress was frequently generated in rice leaves exposed to high temperature. Two-dimensional electrophoresis (2-DE) was used to analyze heat-responsive proteins. More than 600 protein spots were reproducibly detected on each gel; of these spots, 20 were up-regulated, and 7 were down-regulated. A total of 24 proteins and protein species were successfully identified by MALDI-TOF/TOF MS. These proteins and protein species were found to be primarily small heat shock proteins (58%) as well as proteins involved in RNA processing (17%), photosynthesis (13%), chlorophyll biosynthetic processes (4%), protein degradation (4%) and defense (4%). Using 2-DE Western blot analysis, we confirmed the identities of the cytosolic class II small heat shock protein (sHSPs-CII) identified by MS. The expression levels of four different proteins [cytosolic class I small heat shock protein (sHSPs-CI), sHSPs-CII, mitochondrial small heat shock protein (sHSPs-MIT), glycine-rich RNA-binding protein (GRP)] were analyzed at the transcriptional level by quantitative real-time PCR. The mRNA levels of three sHSPs correlated with the corresponding protein levels. However, GRP was down-regulated at the beginning of heat stress but then increased substantially to reach a peak after 24 h of heat stress. Our study provides valuable new insight into the responses of P. ternata to heat stress.

[Influence on AM fungi infection rate and medicine quality of Pinellia ternata in condition of three soil impact factors].

OBJECTIVE: To explore the influence on AM fungi infection rate and medicine quality of Pinellia ternate in the condition of three soil impact factors. METHOD: Set the orthogonal test of three factors and levels. Determinate the AM fungi infection rate in early stage of mature & stage of mature of P. ternata, and the water content, water soluble extract, butanedioic acid content and alkaloid content of P. ternata tuber that be harvested also had be determinated. RESULT AND CONCLUSION: With the P levels to 30 mg x kg(-1) and 90 mg x kg(-1), AM fungi infection was the best when mixed inoculated of EM. Microbial agent inoculated played a decisive role in P. ternata growth and physiological activity, secondary influenced factor was P concentration, and the water stress was the minimal impact. Mixed inoculated of AM fungi and EM treatment with the low P levels (30, 90 mg x kg(-1)) proved better effect on enhancing the water extract content, anedioic acid and alkaloid content, while decreasing the water contents of P. ternata tuber.

[Effect of light quality on growth and quality of Pinellia ternata].

Measuring the content of soluble reducing sugar, total sugar, soluble protein, guanosine, alkaloids, and succinic acid of Pinellia ternata tuber were measured by anthrone-sulfuric acid colorimetric method, Coomassie brilliant blue method, RP-HPLC, reverse potentiometric titration, acid dye colorimetry, respectively. The result showed that yellow light could promote the growth and development of P. ternata and increase the content of soluble reducing sugar, total sugar, alkaloids, and succinic acid. Under blue light could promote the content of soluble protein and guanosine. Red and yellow light increased the content of chlorophyll a and chlorophyll b, contrastively blue light reduced the content of chlorophyll a and chlorophyll b. White film through the most uniform spectrum was most conducive to the synthesis of chlorophyll a. As single film, blue film, yellow film were more conducive to the synthesis of chlorophyll a, green film and red film had been relatively beneficial to the synthesis of chlorophyll b. Bulbil formed the largest number and the biggest propagation coefficient of P. ternata under red light showed that it could increase the production of P. ternata under red light.

Novelties in Microthyriaceae (Microthyriales): Two New Asexual Genera with Three New Species from Freshwater Habitats in Guizhou Province, China.

Microthyriaceae is typified by the sexual genus Microthyrium, with eight asexual genera. Three interesting isolates were collected during our investigation of freshwater fungi from the wetlands in Guizhou Province, southwest China. Three new asexual morphs are identified. Phylogenetic analyses using ITS and LSU gene regions revealed the placement of these isolates in Microthyriaceae (Microthyriales, Dothideomycetes). Based on the morphology and phylogenetic evidence, two new asexual genera, Paramirandina and Pseudocorniculariella, and three new species, Pa. aquatica, Pa. cymbiformis, and Ps. guizhouensis, are introduced. Descriptions and illustrations of the new taxa are provided, with a phylogenetic tree of Microthyriales and related taxa.

[In vitro embryo culture of Epimedium wushanense].

OBJECTIVE: To study the in vitro embryo culture of Epimedium wushanense and provide scientific basis for large scale production of tissue culture. METHOD: Cullus and buds were induced from embryo of E. wushanense on a MS medium supplemented with different 2,4-D,6-BA, NAA, IBA. RESULT: The optimal compositions of medium that induced callus and buds from embryo were the MS medium supplemented with 2,4-D 2 mg x L(-1), IBA 2 mg x L(-1) and NAA 0.5 mg x L(-1) and the MS medium supplemented with IBA 2 mg x L(-1) and 6-BA 0.5 mg x L(-1), respectively. The optimum medium for callus differentiation was MS + 6-BA 1 mg x L(-1) + NAA 0.5 mg x L(-1) + IBA 1 mg x L(-1), and MS +6-BA 1.0 mg x L(-1) + NAA 0.5 mg x L(-1) for shoots proliferation. CONCLUSION: Using embryo as explants, the method of induction and culture of E. wushanense was established by the callus and buds, and the embryo of E. wushanense can be quickly propagated.

[Tissue cultivation of tiller buds of Epimedium wushanense].

OBJECTIVE: To established the rapid tissue propagation system of Epimedium wushanense, in order to provide theoretical basis for industrialized seed cultivation. METHOD: Tiller buds E. wushanense were used as explants, with MS, B5, WPM as basic media, and added with different concentrations of plant growth regulators such as 6-BA, NAA and GA3, in order to conduct a systematic study on induction and propagation conditions for tiller buds. RESULT: The suitable method for sterilizing bud was to disinfect with 75% ethanol for 30 s, and then treated with 0.1% HgCl2 for (4 + 2) min for consecutively twice, which could control the pollution rate below 5% and the survival rate above 75%. The optimal medium for bud induction was WPM + 6-BA 2.0 mg x L(-1) + NAA 0.1 mg x L(-1) + GA3 0.5 mg x L(-1), with the induction rate of 75.5%; meanwhile, the basic medium and 6-BA showed significant effect on the induction rate. The propagation medium suitable for buds was MS +6-BA 2.0 mg x L(-1) + NAA 0.5 mg x L(-1), with the propagation rate of 3.3. The optimal growth of rooting medium was 1/2 WPM + IBA 0.5 mg x L(-1) + activated carbon which (0.05%), with the rooting rate of 90%, three to six strong seedlings in each plant. CONCLUSION: The disinfection method suitable for tiller buds and the medium combination suitable for induction, propagation and rooting of adventitious buds are screened out to establish the rapid cultivation system for tiller buds of E. wushanense.

Achog1 is required for the asexual sporulation, stress responses and pigmentation of Aspergillus cristatus.

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea; hypotonic conditions only induce its sexual development to produce ascospores, while hypertonic conditions only induce its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the role of Achog1, which is homologous to hog1 from Saccharomyces cerevisiae, in sporulation, different kinds of stress responses and pigment production was investigated. Deletion mutants of Achog1 were obtained by homologous recombination. Phenotypic observations showed that the time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in the deletion mutants of Achog1 in hypertonic media, indicating that Achog1 plays a positive role in asexual development. Stress sensitivity tests showed that ΔAchog1 strains were sensitive to hyperosmolarity, and the order of the sensitivity of ΔAchog1 to different osmotic regulators was 3 M sucrose >3 M NaCl >3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. pH sensitivity tests indicated that Achog1 inhibited the growth of A. cristatus under alkaline stress. Additionally, pigmentation was decreased in the Achog1 deletion mutants compared with the WT. All the above developmental defects were reversed by the reintroduction of the Achog1 gene in ΔAchog1. Pull-down and LC-MS/MS analysis showed that the expression levels of proteins interacting with Achog1 were significantly different under low and high osmotic stress, and proteins related to conidial development were present only in the cultures treated with hyperosmotic stress. Transcription profiling data showed that Achog1 suppressed the expression of several genes related to asexual development, osmotic and oxidative stress resistance. On the basis of gene knockout, pull-down mass spectrometry and RNA-seq analyses, a regulatory pathway for Achog1 was roughly identified in A. cristatus.

Curvicladiellapaphiopedili sp. nov. (Hypocreales, Nectriaceae), a new species of orchid (Paphiopedilum sp.) from Guizhou, China.

Background: An asexual fungus, collected from diseased leaves of Paphiopedilum sp. from Guizhou Province, China, and based on the phylogenetic analyses and morphological characters, it was identified as a new species in Curvicladiella. The genus Curvicladiella are recorded for the first time for China. New information: The morphology of Curvicladiellapaphiopedili sp. nov. is characterised by penicillate conidiophores with a stipe, dull, tapering towards the apex, the curved stipe extension and cylindrical conidia. In the phylogenetic analyses of combined cmdA, his3, ITS, LSU, tef1 and tub2 sequence data, this taxon was clustered as sister to Curvicladiellacignea within Nectriaceae.