A new polyporoid fungus, Physisporinus sulphureus, is described from Singapore, Southeast Asia. It is characterized by annual, resupinate and soft basidiocarps with sulphurous hymenophores when fresh and olivaceous buff to honey-yellow when dry, a monomitic hyphal system, simply septate generative hyphae, the presence of hyphoid cystidia and subulate cystidioles, and subglobose to globose basidiospores with a guttule. Molecular phylogeny inferred from ITS and LSU sequences data strongly supported P. sulphureus as a distinctive species belonging to Physisporinus. The new species is described and illustrated in this paper.
Five cercosporoid hyphomycetes on Sterculiaceae in China are reported, of which, two are new species, Pseudocercospora firmianae and P. firmianicola, and one is new record for China, P. corchorifoliae on Firmiana simplex. Latin descriptions, illustrations and discussions are provided. Examined specimens are deposited in HMAS.
The genetic diversity of 60 strains of Puccinia polysora collected from Shandong, Jiangsu, Zhejiang, Henan and Hainan provinces from 2014 to 2016 was analyzed by using eleven pair of polymorphic ISSR primers. The results indicated that, the genetic diversity of the strains collected from Shandong Province was abundant, and its polymorphic percentage ( P) was 96.34%. The genetic diversity of the strains was highly similar to that of the strains collected from Jiangsu, Zhejiang and Henan provinces, and the genetic distance was relatively high. The strains collected from Shandong Province showed the highest genetic similarity coefficient (GS=0.7829) and the closest genetic distance (GD=0.2447) as compared to those collected from Jiangsu Province; however, the strains from Shandong showed the lowest genetic similarity coefficient (GS=0.0148) and the farthest genetic distance (GD=4.2999) as compared to those of collected from Hainan Province. At the level of similarity coefficient 0.74, the 60 strains were classified into three groups and several subgroups, and there were obvious differences in annual and geographical distribution between the groups and subgroups. Gst value of the strains collected in 2015 was 0.8694, and that of the strains collected both in 2015 and in 2016 was 0.4562, indicating that the genetic variation of the pathogen was reflected not only in different regions, but also in different years. Based on ISSR marker analysis, the authors believed that the primary infection sources of the disease occurred in Shandong Province were not from Hainan Province, but probably from the pathogen overwintering region, such as Philippines or Taiwan.
Based on a gene knock-in strategy, the reporter gene GFP (or mCherry) was fused with a histone H1 gene and the cassettes were transformed and incorporated into the genome of Colletotrichum fructicola for in-fusion expression and nuclear labeling. Based on the obtained strains, live image of nuclear behavior was possible for various fungal structures such as conidium, vegetative hyphae, appressorium, and infectious hyphae. C. fructicola contains sexually compatible ‘plus’ and ‘minus’ strains, GFP-labeled ‘plus’ strain and mCherry-labeled ‘minus’ strain formed a ‘mating line’ at the contact zone. Both mCherry-labeled and GFP-labeled ascospores could be observed within the same ascus at the ‘mating line’, demonstrating crossing events between the ‘plus’ and ‘minus’ strains. The cell walls of ‘mating line’ perithecia expressed mCherry, demonstrating their ‘minus’ strain derivation. The nuclear-labeled strains reported here allow for convenient and direct observation of nuclear behaviors, and will be important materials for studying cell cycle regulations and sexual reproductions of C. fructicola.
Verticillium dahliae, a typical soilborne fungus, infects over 400 plant species and causes huge economic loss. Microsclerotia can survive for 14 years in the soil as dormancy structures and serve as the primary inoculum on many hosts. According to the whole genome-wide expression profiles on the germination of microsclerotia, the VDAG_03943 gene, coding cyclopentanone 1,2-monooxygenase, was most highly expressed by the fungus during the germination process. In the present study, we cloned the sequence and characterized the function of VDAG_03943. Sequence analysis showed that VDAG_03943, named VdCPMO, has a full length of 1 929bp. The cDNA coding region of VdCPMO contains 1 668bp of nucleotides that encode a protein consisted of 555 amino acids. The results displayed that deletion mutant strains exhibited several developmental defects including slower growth of colony, reduced germination rate of microsclerotia, and reduced average length of germ tube as compared with the wild type strain. There were no significant differences between the complementary mutant strain and wild type JY strain. Furthermore, the microsclerotia of deletion mutant strains lost the pathogenicity to cotton.
Alternative splicing is an important way to regulate gene expression. In this study, four alternative splicing variants ( exg2V1, exg2V2, exg2V3 and exg2V4) of Volvariella volvacea exo-β-1,3-glucanase gene exg2 were identified by RT-PCR. The types of alternative splicing variants were as follows: the 7th intron retention in exg2V1, the 11th intron retention and the last 58bp nucleotides of the 12th exon excision in exg2V2, the 7th and 17th introns’ retention in exg2V3, and the 2nd, 9th, and 17th introns’ retention in exg2V4. There was premature translation termination codon in all of four alternative splicing variants, resulting in the inclusion of an incomplete domain in the predicted amino acid sequence. The results of quantitative PCR showed that the expression levels of the four alternative splicing variants were lower than that of the standard exg2 transcript in all five developmental stages of V. volvacea. However, there were still some differential expression among the developmental stages and tissues for the four alternative splicing variants. It is deduced that all of four alternative splicing variants of the V. volvacea exg2 gene belong to nonsense-mediated mRNA decay, by this way to achieve the precise regulation of standard exg2 gene transcript.
The metabolites of mycelial samples of ΔveA mutant and wild-type strain E4 of Aspergillus cristatus with 48 hour incubation period were identified by gas chromatography-mass spectrometry (GC-MS). A total of 99 metabolites were identified, including 41 significantly differential metabolites. Among them, 20 and 21 metabolites were much increased in the wild-type E4 and ΔveA, respectively. Statistical analysis revealed there were 623 individual metabolite having correlation, of which 313 were positive and 310 negative. These metabolites include organic acids, amino acids, carbohydrates, alcohols and oxylipin, while organic acids and amino acids predominate. This result lays the foundation for further study on the correlation between metabolite and sporulation in A. cristatus.
The PEG mediated method for genetic transformation of Epicoccum nigrum was carried out. The sensitivity assessment of E. nigrum to hygromycin B showed that the lowest selection concentration was 50mg/L. The optimum condition for preparing protoplasts and transformation was set up by using 2% lysing enzyme in 0.7mol/L NaCl to digest the fresh mycelia at 30°C for 2h to produce an efficient yield of protoplasts. After washing by MTC buffer twice, the protoplasts were incubated with expression plasmid pYF11-hph and mixed with RM medium. Verifications of the putative transformants by hygromycin B seletion and PCR analysis indicated that the hph gene has successfully transformed into the protoplast of E. nigrum. The stable PEG-mediated genetic transformation system provides an essential technology for studying the metabolic pathways, molecular mechanisms, and constructing engineering strains.
The difference of Inonotus hispidus and Sanghuangporus spp . was revealed at molecular level. The active constituents of Inonotus hispidus against breast cancer cell line MDA-MB-231 were screened and their active ingredients were analysed. The phylogenetic tree and split network based on rDNA ITS sequence was used to analyse the difference of Inonotus hispidus and Sanghuangporus spp. MTT method was used to detect the anti-breast cancer activity of different extracts of Inonotus hispidus fermentation broth. The effect of extraction fractions on cancer cell growth was detected by morphological observation. LC-MS/MS was used to analyze the active constituents of the extracts. The phylogenetic tree showed that Inonotus hispidus and Sanghuangporus spp. had certain genetic differences. The GC content of Inonotus hispidus were lower than that of Sanghuangporus spp. and the split network analysis could distinguish Inonotus hispidus from Sanghuangporus spp. N-butanol extract of Inonotus hispidus fermentation broth had the strongest inhibitory effect on the growth of MDA-MB-231 cells and the cell proliferation was inhibited in a dose and time dependent manner ( P<0.05) and the IC50 value for 24 hours was 245.44μg/mL. Cell morphology observation showed that the effect of n-butanol extract at 250μg/mL concentration on the apoptosis of MDA-MB-231 cells increased with the prolong of treatment time. LC-MS/MS data had identified two chemical components, and they were recognized from Inonotus hispidus for the first time. The n-butanol extract of Inonotus hispidus fermentation broth could be considered having anti-breast cancer activity.
Determination of tumor inhibition rate and spleen and thymus index, and observation of tumor slices and the changes of interferon-γ (IFN-γ), interleukin-2 (IL-2), vascular endothelial growth factor (VEGF), catalase (CAT), glutathione-peroxidase (GSH-PX) and superoxide dismutase (SOD) in serum were carried out to analyze the anti-tumor activities and immune regulation effects of Grifola frondosa fruiting body coarse powder and submicron powder on Hep-A-22 tumor-bearing mice. The results showed that the anti-tumor remedy of high dose submicron powder (1 500mg/kg) experimental group was of the best effect showing significant difference ( P<0.01) as compared with the model experimental group. Increased necrotic area of tumor was observed by HE staining. The content of IFN-γ, IL-2, VEGF, GSH-PX and SOD in serum of the treated group were significantly increased, showing a correlation with the tumor inhibition rate.
The natural products of Pestalotiopsis has become a research hotspot as they can produce diverse secondary metabolites including taxol, the antitumor agent. UPLC-Q-TOF-MS diagram analysis of the metabolites from endolichenic Pestalotiopsis sp. revealed that there were two metabolites with unique molecular weight. Further isolation with different chromatography approaches of the crude extract afforded two compounds with complex structural features. The structures of the compounds were elucidated to be a known torreyanic acid derivative (1) and a new analogue named pestalotiopsin (2) by extensive NMR experiments including 1H, 13C, HMQC, HMBC, ROESY and HRESIMS data. The absolute configuration of the new compound (2) was determined by comparing the CD spectrum with known analogue (1). Compounds 1 and 2 exhibited cytotoxic activity against human leukemia cell line K562 in vitro, with IC50 values of 25.2 and 32.1µmol/L, respectively.
In the desert lichen Endocarpon pusillum can not be detected any secondary metabolite. But, its mycobiont genome contains fourteen PKS genes and two NRPS genes. Through the activation of culture in vermiculite medium, three secondary metabolites have been obtained from the mycobiont, including a new isoindolin-1-one compound (endocarpin A), a new naphthoquinone (endocarpin B) and lamellicolic anhydride. Other three demagnesium chlorophyll compounds were isolated from its phycobiont Diplosphaera chodatii. They are phaeophorbide b, 13- epi-phaeophorbide a and phaeophorbide a. Phaeophorbide b owned medium antioxidant activity based on the ability to scavenge DPPH free radicals in vitro.
Based on high-throughput sequencing technology, species diversity of the endophytic fungi from Robinia pseudoacacia in Huaxi District, Guiyang City, Guizhou Province were analyzed. The results showed that there were 43 942 valid sequences. Based on cluster similarity analysis 81 OTUs were obtained, belonging to 4 phyla, 13 classes, 27 orders, 45 families, and 58 genera. Among these fungi, the dominant was the unclassified genus, and its relative abundance was 81.11% of the total isolates. The next was Fusarium (11.81%), Gloniopsis (1.38%) and Clonostachys (0.72%), while the relative abundances of more than 50 genera were less than 0.5%. The Shannon-Wiener’s diversity index and the Simpson’s index of the endophytic fungi in the samples were 0.6835 and 0.7071 respectively. FUNGuild software platform analysis displayed that endophytic fungi of Robinia pseudoacacia contained the following functions, groups: plant pathogen, animal pathogen, endophyte, fungal parasite, lichen parasite, dung saprotroph, undefined saprotroph fungi and wood saprotroph.
Three species of Pucciniaceae collected from Inner Mongolia, Puccinia albescens on Adoxa moschatellina, Puccinia stipina on Stipa sareptana var. krylovii, and Uromyces gageae on Lloydia triflora, are reported as new to China. Morphological descriptions, illustrations and ITS sequences are provided based on Chinese collections. The collections are deposited in the Mycological Herbarium of Chifeng University, Chifeng, Inner Mongolia, China (CFSZ), and Herbarium Mycologicum Academiae Sinicae, Beijing, China (HMAS).
