Rosellichalasins A-H, cytotoxic cytochalasans from the endophytic fungus Rosellinia sp. Glinf021.

Eight new cytochalasans rosellichalasins A-H (1-8), as well as two new shunt metabolites rosellinins A (9) and B (10) before intramolecular Diels-Alder cycloaddition reaction in cytochalasan biosynthesis, along with nine known cytochalsans (11-19) were isolated from the endophytic fungus Rosellinia sp. Glinf021, which was derived from the medicinal plant Glycyrrhiza inflata. Their structures were characterized by extensive analysis of 1D and 2D NMR as well as HRESIMS spectra and quantum chemical ECD calculations. The cytotoxic activities of these compounds were evaluated against four human cancer cell lines including HCT116, MDA-MB-231, BGC823, and PANC-1 with IC50 values ranging from 0.5 to 58.2 µM.

Pestalotiopsis-Like Species Causing Gray Blight Disease on Camellia sinensis in China.

Gray blight of tea, caused by several Pestalotiopsis-like species, is one of the most destructive foliar diseases in tea cultivation yet the characteristics of these pathogens have not been confirmed until now. With morphological and multigene phylogenetic analyses, we have identified the gray blight fungi as Pseudopestalotiopsis camelliae-sinensis, Neopestalotiopsis clavispora, and Pestalotiopsis camelliae. Phylogenetic analyses derived from the combined internal transcribed spacer, ß-tubulin, and translation elongation factor 1-α gene regions successfully resolved most of the Pestalotiopsis-like species used in this study with high bootstrap supports and revealed three major clusters representing these three species. Differences in colony appearance and conidia morphology (shape, size, septation, color and length of median cells, and length and number of apical and basal appendages) were consistent with the phylogenetic grouping. Pathogenicity tests validated that all three species isolated from tea leaves were causal agents of gray blight disease on tea plant (Camellia sinensis). This is the first description of the characteristics of the three species Pseudopestalotiopsis camelliae-sinensis, N. clavispora, and Pestalotiopsis camelliae as causal agents of tea gray blight disease in China.

Morphological and Molecular Analysis Identifies the Associated Fungus ("Xianghui") of the Medicinal White Jelly Mushroom, Tremella fuciformis, as Annulohypoxylon stygium.

White jelly mushroom, Tremella fuciformis, is a popular edible mushroom with interesting medicinal properties (e.g., immunostimulating, antidiabetic). The formation of T. fuciformis basidiomes is highly dependent on the presence of a specific host fungus, both in nature and for industrial production. This host has traditionally been indicated as "Xianghui" in China, yet which or how many fungal species Xianghui comprises is unclear, with various authorities claiming different species. At present, Annulohypoxilon archeri is generally assumed to be the main Xianghui species, but this has not yet been confirmed experimentally. The implementation of older, premolecular-based research data (i.e., morphological) with present, sequence-based data to solve the identity remains confusing and studies addressing both identification methods in combination are lacking. The unclear identity of Xianghui is a major obstacle for further understanding of the important relationship between the host(s) and T. fuciformis. In this study, we collected a wild specimen of T. fuciformis together with several nearby stroma of Xianghui, cocultivated T. fuciformis with the Xianghui isolates, and observed basidiome formation. Internal transcribed spacer (ITS) sequence analysis showed that all Xianghui spore isolates belonged to the same species and both morphological analysis of sexual stages and ITS ß-tubulin and actin gene sequences of the Xianghui specified it as Annulohypoxylon stygium. The ITS sequences of the newly identified Xianghui further closely matched those of the Xianghui strains used in the mushroom industry, showing that wild and culture spawn Xianghui in China consist of A. stygium. In contrast with previous conclusions, A. stygium, and not A. archeris, seems to be the preferred host of T. fuciformis.

A Bilobalide-Producing Endophytic Fungus, Pestalotiopsis uvicola from Medicinal Plant Ginkgo biloba.

For screening bilobalide (BB)-producing endophytic fungi from medicinal plant Ginkgo biloba, a total of 57 fungal isolates were isolated from the internal stem, root, leaf, and bark of the plant G. biloba. Fermentation processes using BB-producing fungi other than G. biloba may become a novel way to produce BB, which is a terpene trilactones exhibiting neuroprotective effects. In this study, a BB-producing endophytic fungal strain GZUYX13 was isolated from the leaves of G. biloba grown in the campus of Guizhou University, Guiyang city, Guizhou province, China. The strain produced BB when grown in potato dextrose liquid medium. The amount of BB produced by this endophytic fungus was quantified to be 106 µg/L via high-performance liquid chromatography (HPLC), substantially lower than that produced by the host tissue. The fungal BB which was analyzed by thin layer chromatography (TLC) and HPLC was proven to be identical to authentic BB. The strain GZUYX13 was identified as Pestalotiopsis uvicola via morphology and ITS rDNA phylogeny. To the best of our knowledge, this is the first report concerning the isolation and identification of endophytic BB-producing Pestalotiopsis spp. from the host plant, which further proved that endophytic fungi have the potential to produce bioactive compounds.

Hypoxylon pulicicidum sp. nov. (Ascomycota, Xylariales), a pantropical insecticide-producing endophyte.

BACKGROUND: Nodulisporic acids (NAs) are indole diterpene fungal metabolites exhibiting potent systemic efficacy against blood-feeding arthropods, e.g., bedbugs, fleas and ticks, via binding to arthropod specific glutamate-gated chloride channels. Intensive medicinal chemistry efforts employing a nodulisporic acid A template have led to the development of N-tert-butyl nodulisporamide as a product candidate for a once monthly treatment of fleas and ticks on companion animals. The source of the NAs is a monophyletic lineage of asexual endophytic fungal strains that is widely distributed in the tropics, tentatively identified as a Nodulisporium species and hypothesized to be the asexual state of a Hypoxylon species. METHODS AND RESULTS: Inferences from GenBank sequences indicated that multiple researchers have encountered similar Nodulisporium endophytes in tropical plants and in air samples. Ascomata-derived cultures from a wood-inhabiting fungus, from Martinique and closely resembling Hypoxylon investiens, belonged to the same monophyletic clade as the NAs-producing endophytes. The hypothesis that the Martinique Hypoxylon collections were the sexual state of the NAs-producing endophytes was tested by mass spectrometric analysis of NAs, multi-gene phylogenetic analysis, and phenotypic comparisons of the conidial states. We established that the Martinique Hypoxylon strains produced an ample spectrum of NAs and were conspecific with the pantropical Nodulisporium endophytes, yet were distinct from H. investiens. A new species, H. pulicicidum, is proposed to accommodate this widespread organism. CONCLUSIONS AND SIGNIFICANCE: Knowledge of the life cycle of H. pulicicidum will facilitate an understanding of the role of insecticidal compounds produced by the fungus, the significance of its infections in living plants and how it colonizes dead wood. The case of H. pulicicidum exemplifies how life cycle studies can consolidate disparate observations of a fungal organism, whether from environmental sequences, vegetative mycelia or field specimens, resulting in holistic species concepts critical to the assessment of the dimensions of fungal diversity.

Effect of water activity on the production of volatile organic compounds by Muscodor albus and their effect on three pathogens in stored potato.

Muscodor albus (Xylariaceae, Ascomycetes) isolate CZ-620 produces antimicrobial volatile organic compounds (VOC), which appear to have potential for the control of various postharvest diseases. The effect of water activity (Aw) on the production of VOC by M. albus culture, and their inhibitory effects on the growth of three pathogens of potato tuber (Fusarium sambucinum, Helminthosporium solani, and Pectobacterium atrosepticum) and the development of diseases caused by the three pathogens (dry rot, silver scurf, and bacterial soft rot, respectively) were investigated. Rye grain culture of the fungus produced six alcohols, three aldehydes, five acids or esters, and two terpenoids. The most abundant VOC were: isobutyric acid; bulnesene, a sesquiterpene; an unidentified terpene; 2 and 3-methyl-1-butanol; and ethanol. However, the level of each of those VOC varied with Aw of the culture. Emission activity occurred mainly at Aw above 0.75 and high emission of most VOC occurred only at Aw above 0.90. The aldehydes (2-methyl-propanal and 3-methyl-butanal) were the only VOC produced in quantities below an Aw of 0.90. An Aw value of 0.96 favored maximum emission of acids, esters, and terpenoids. There was a higher production of alcohols and a decrease in aldehydes with increase in Aw. Isobutyric acid, which has been the main M. albus VOC monitored in previous studies as an indicator of antifungal activity, had a rather narrow optimum, peaking at Aw of 0.96 and declining sharply above 0.98. Results showed that substrate Aw affects the production dynamics of each group of VOC by the fungus, and suggest that VOC production can be prolonged by maintaining M. albus culture at a constant optimum Aw. The VOC was inhibitory to F. sambucinum, H. solani, and P. atrosepticum; and biofumigation with M. albus significantly reduced dry rot and soft rot development, and completely controlled silver scurf in inoculated tubers incubated at both 8°C and 22°C. The results show that Aw of grain culture affects the production of VOC by M. albus; and that the VOC inhibit the growth of the tested pathogens and the diseases caused by them in potato tubers.

Bioprospecting highly diverse endophytic Pestalotiopsis spp. with antibacterial properties from Maytenus ilicifolia, a medicinal plant from Brazil.

Thirteen endophytic fungal strains of the genus Pestalotiopsis were isolated from the medicinal plant Maytenus ilicifolia Mart. ex. Reiss (commonly known as "espinheira santa") and their antimicrobial properties were investigated. Two isolates were successful in inhibiting the growth of the tested microorganisms (Escherichia coli, Klebsiella pneumoniae, Micrococcus luteus, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA)) using the technique of bioautographic thin-layer chromatography (TLC) agar overlay assay. An analysis based on a polyphasic approach integrating taxonomic information, morphological traits, RAPD markers, and the sequencing of the ITS1-5.8S-ITS2 of the rDNA led to the assignment of the isolates as belonging to the species Pestalotiopsis microspora, Pestalotiopsis vismiae, and Pestalotiopsis leucothoes. Therefore, the present study presents a new approach to the study of endophytic fungi of the genus Pestalotiopsis.