Two transcription factors cooperatively regulate DHN melanin biosynthesis and development in Pestalotiopsis fici.

Fungal 1,8-dihydroxynaphthalene (DHN) melanin plays important roles in UV protection, oxidative stress and pathogenesis. However, knowledge of the regulatory mechanisms of its biosynthesis is limited. Previous studies showed two transcription factors, PfmaF and PfmaH, located in the DHN melanin biosynthetic gene cluster (Pfma) in Pestalotiopsis fici. In this study, deletion of PfmaH resulted in loss of melanin and affected conidia cell wall integrity. Specifically, PfmaH directly regulates the expression of scytalone dehydratase, which catalyzes the transition of scytalone to T3 HN. However, PfmaF disruption using CRISPR/Cas9 system affected neither DHN melanin distribution nor conidia cell wall integrity in P. fici. Unexpectedly, overexpression of PfmaF leads to heavy pigment accumulation in P. fici hyphae. Transcriptome and qRT-PCR analyses provide insight into the roles of PfmaF and PfmaH in DHN melanin regulation. PfmaH, as a pathway specific regulator, mainly regulates melanin biosynthesis that contributes to cell wall development. Furthermore, PfmaF functions as a broad regulator to stimulate PfmaH expression in melanin production, secondary metabolism as well as fungal development.

A new regulator RsdA mediating fungal secondary metabolism has a detrimental impact on asexual development in Pestalotiopsis fici.

Secondary metabolite (SM) production and development are correlated processes in fungi that are often coordinated by pleiotropic regulators. The eukaryotic regulators are critical players in mediating SM production related to fungal development, yet little data are available to support this hypothesis. In this study, a global regulator, RsdA (regulation of secondary metabolism and development), was identified through genome-wide analysis and deletion of the regulator gene in the endophytic fungus Pestalotiopsis fici. Here, we established that RsdA regulation of SMs is accompanied by the repression of asexual development. Deletion of rsdA significantly reduces not only asexual development, resulting in low sporulation and abnormal conidia, but also the major SM production, while remarkably increasing the melanin production. Overproduction of melanin leads to the formation of unusual, heavily pigmented hyphae. Transcriptome analysis data provide the evidence that RsdA globally regulates genes involved in secondary metabolism and asexual development. Double deletion of rsdA and the melanin polyketide synthase gene PfmaE confirm that RsdA regulation of asexual development is independent of the melanin biosynthetic pathway. Finally, our results demonstrate that RsdA can be used for the discovery of secondary metabolites in filamentous fungi.

Diverse members of the Xylariales lack canonical mating-type regions.

A survey of genomes reported here for 10 isolates of Monosporascus species and an additional 25 genomes from other members of the Xylariales (representing 15 genera) available in public databases indicated that genes typically associated with MAT1-1 (mat A) or MAT1-2 (mat a) mating types are absent or have diverged greatly relative to counterparts in other Pezizomycotina. This was particularly surprising for isolates known to be homothallic, given that homothallic members of the Pezizomycotina typically possess a MAT1-1-1 (mat A-1) gene and one or both of two other closely-linked mating-type genes, MAT1-1-2 (mat A-2) and MAT1-1-3 (mat A-3), in addition to MAT1-2-1 (mat a-1). We failed to detect candidate genes for either MAT1-1-1 or MAT1-1-2 in any member of the Xylariales. Genes related to MAT1-2-1 and MAT1-1-3 are present in the genomes examined, but most appear to be orthologs of MATA_HMG (high-mobility group) genes with non-mating-type functions rather than orthologs of mating-type genes. Several MATA_HMG genes were found in genome positions that suggest they are derived from mating-type genes, but these genes are highly divergent relative to known MAT1-2-1 and MAT1-1-3 genes. The genomes examined represent substantial diversity within the order and include M. cannonballus, M. ibericus, Xylaria hypoxylon, X. striata, Daldinia eschscholzii, Eutypa lata, Rosellinia necatrix, Microdochium bolleyi and several others. We employed a number of avenues to search for homologs, including multiple BLAST approaches and examination of annotated genes adjacent to genes known to flank mating regions in other members of the Ascomycota. The results suggest that the mating regions have been lost from, or altered dramatically in, the Xylariales genomes examined and that mating and sexual development in these fungi are controlled differently than has been reported for members of the Pezizomycotina studied to date.

Response of the Biocontrol Agent Pseudomonas pseudoalcaligenes AVO110 to Rosellinia necatrix Exudate.

The rhizobacterium Pseudomonas pseudoalcaligenes AVO110, isolated by the enrichment of competitive avocado root tip colonizers, controls avocado white root rot disease caused by Rosellinia necatrix Here, we applied signature-tagged mutagenesis (STM) during the growth and survival of AVO110 in fungal exudate-containing medium with the goal of identifying the molecular mechanisms linked to the interaction of this bacterium with R. necatrix A total of 26 STM mutants outcompeted by the parental strain in fungal exudate, but not in rich medium, were selected and named growth-attenuated mutants (GAMs). Twenty-one genes were identified as being required for this bacterial-fungal interaction, including membrane transporters, transcriptional regulators, and genes related to the metabolism of hydrocarbons, amino acids, fatty acids, and aromatic compounds. The bacterial traits identified here that are involved in the colonization of fungal hyphae include proteins involved in membrane maintenance (a dynamin-like protein and ColS) or cyclic-di-GMP signaling and chemotaxis. In addition, genes encoding a DNA helicase (recB) and a regulator of alginate production (algQ) were identified as being required for efficient colonization of the avocado rhizosphere.IMPORTANCE Diseases associated with fungal root invasion cause a significant loss of fruit tree production worldwide. The bacterium Pseudomonas pseudoalcaligenes AVO110 controls avocado white root rot disease caused by Rosellinia necatrix by using mechanisms involving competition for nutrients and niches. Here, a functional genomics approach was conducted to identify the bacterial traits involved in the interaction with this fungal pathogen. Our results contribute to a better understanding of the multitrophic interactions established among bacterial biocontrol agents, the plant rhizosphere, and the mycelia of soilborne pathogens.

Antifungal Volatile Organic Compounds from the Endophyte Nodulisporium sp. Strain GS4d2II1a: a Qualitative Change in the Intraspecific and Interspecific Interactions with Pythium aphanidermatum.

This study demonstrates volatile organic compounds (VOCs) production as one of the defense mechanisms of the antagonistic endophyte Nodulisporium sp. GS4d2II1a, and the volatile changes in two times of the fungal growth; and, as result of its intra and interspecific interactions with the plant pathogen Pythium aphanidermatum. The antifungal activity of the volatile and diffusible metabolites was evaluated by means of three types of antagonism bioassays and by organic extract agar dilution. VOCs were obtained by gas chromatography coupled to mass spectrometry from 3- and 5-day Nodulisporium sp. cultures, as well as from its interspecific in vitro antagonistic interaction with the oomycete P. aphanidermatum, and its intraspecific Nodulisporium sp.-Nodulisporium sp. interaction. The GS4d2II1a strain completely inhibited the growth of two fungi and seven oomycetes by replacing their mycelia in simple antagonism bioassays and by producing in vitro volatile and diffusible metabolites that acted synergistically in multiple antagonism bioassays. Additionally, VOCs inhibited the growth of three oomycetes and one fungus in antagonism bioassays using divided plates. A total of 70 VOCs were detected, mainly including mono and sesquiterpenes, especially eucalyptol and limonene. Multiple correspondence analysis revealed four different volatile profiles, showing that volatiles changed with the fungus age and its intra and interspecific interactions. The metabolites produced by Nodulisporium sp. GS4d2II1a could be useful for biological control of fungal and oomycetes plant pathogens of economically important crops.

Interactions between head blight pathogens: consequences for disease development and toxin production in wheat spikes.

Head blight (HB) is one of the most damaging diseases on wheat, inducing significant yield losses and toxin accumulation in grains. Fungal pathogens responsible for HB include the genus Microdochium, with two species, and the toxin producer genus Fusarium, with several species. Field studies and surveys show that two or more species can coexist within a same field and coinfect the same plant or the same spike. In the current study, we investigated how the concomitant presence of F. graminearum and another of the HB complex species influences the spike colonization and the toxin production by the fungi. To study these interactions, 17 well-characterized isolates representing five species were inoculated alone or in pairs on wheat spikes in greenhouse and field experiments. The fungal DNA in the grains was estimated by quantitative PCR and toxin contents (deoxynivalenol and nivalenol) by ultraperformance liquid chromatography-UV detection-tandem mass spectrometry. The responses of the different isolates to the presence of a competitor were variable and isolate specific more than species specific. The development of the most aggressive isolates was either unchanged or a slightly increased, while the development of the less aggressive isolates was reduced. The main outcome of the study was that no trend of increased toxin production was observed in coinoculations compared to single inoculations. On the contrary, the amount of toxin produced was often lower than expected in coinoculations. We thus conclude against the hypothesis that the co-occurrence of several HB-causing species in the same field might aggravate the risk linked to fusarium toxins in wheat production.

Biosynthesis of integric acid isolated from the wood-decay fungus Xylaria feejeensis 2FB-PPM08M.

The biosynthesis of integric acid, a secondary metabolite of the wood-decay fungus Xylaria feejeensis strain 2FB-PPM08M, has been studied. Labeling experiments using [1-(13)C], [2-(13)C] and [1,2-(13)C2] acetate and L-methionine (methyl-(13)C) were separately performed with fungal culture. The labeling patterns of these metabolites indicated the same origin, and determined that integric acid was formed through the condensation of a sesquiterpene and a polyketide. These experiments showed that side chain of compounds would be synthesized by the polyketide pathway, while the ring carbon indicated the biosynthesis of compounds via the mevalonate pathway.

Xyloketal B suppresses glioblastoma cell proliferation and migration in vitro through inhibiting TRPM7-regulated PI3K/Akt and MEK/ERK signaling pathways.

Glioblastoma, the most common and aggressive type of brain tumors, has devastatingly proliferative and invasive characteristics. The need for finding a novel and specific drug target is urgent as the current approaches have limited therapeutic effects in treating glioblastoma. Xyloketal B is a marine compound obtained from mangrove fungus Xylaria sp. (No. 2508) from the South China Sea, and has displayed antioxidant activity and protective effects on endothelial and neuronal oxidative injuries. In this study, we used a glioblastoma U251 cell line to (1) explore the effects of xyloketal B on cell viability, proliferation, and migration; and (2) investigate the underlying molecular mechanisms and signaling pathways. MTT assay, colony formation, wound healing, western blot, and patch clamp techniques were employed. We found that xyloketal B reduced cell viability, proliferation, and migration of U251 cells. In addition, xyloketal B decreased p-Akt and p-ERK1/2 protein expressions. Furthermore, xyloketal B blocked TRPM7 currents in HEK-293 cells overexpressing TRPM7. These effects were confirmed by using a TRPM7 inhibitor, carvacrol, in a parallel experiment. Our findings indicate that TRPM7-regulated PI3K/Akt and MEK/ERK signaling is involved in anti-proliferation and migration effects of xyloketal B on U251 cells, providing in vitro evidence for the marine compound xyloketal B to be a potential drug for treating glioblastoma.

Microdochium paspali, a new species causing seashore paspalum disease in southern China.

A new species of Microdochium was identified as the causal agent of leaf blight of seashore paspalum (Paspalum vaginatum), a turf grass widely used in tropical and subtropical golf courses. In 2010 foliar necrosis and canopy thinning were observed on 11 surveyed golf courses in Hainan province, China, especially on fairways and putting greens. The infected leaves initially appeared water-soaked and dark green, rapidly faded to yellow or became chlorotic and quickly died, resulting in a sparse appearance in infected areas, leading to the disease name "sparse leaf patch." Isolates with rich and light pink to yellow mycelia and salmon-colored pionnotes were cultured from diseased turf foliage. Pathogenicity was demonstrated by inoculating these isolates onto "seaspray" seashore paspalum. Phylogenetic analysis based on the nuc rDNA internal transcribed spacer 1-5.8S-internal transcribed spacer 2 region (ITS), translation elongation factor 1-α (TEF1-α) and ß-tubulin (BenA) indicated these isolates formed as a distinct clade within Microdochium (Xylariales). Further microscopic examination demonstrated that the species was morphologically distinct from three similar species of Microdochium. The name Microdochium paspali sp. nov. is proposed for this novel fungal pathogen.

Molecular phylogeny of Ascotricha, including two new marine algae-associated species.

Phylogenetic analyses based on a broad taxonomic sampling of Ascotricha were conducted using the sequences of nuc rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S rDNA (ITS), partial nuc 18S rDNA (18S) and partial ß-tubulin gene (TUB2). Hypoxyloid Xylariaceae and xylarioid Xylariaceae were inferred as two distinct lineages in the Xylariaceae in the combined ITS-TUB2 phylogeny. Within xylarioid Xylariaceae species of Ascotricha form a monophyletic group. Two new marine algae-associated fungi, Ascotricha longipila and A. parvispora, are described on the basis of morphological and molecular characters and the combination, A. sinuosa, is proposed. A synopsis of the morphological characters and a dichotomous key to Ascotricha species are provided.

The genus Cryptosphaeria in the western United States: taxonomy, multilocus phylogeny and a new species, C. multicontinentalis.

This study investigates the diversity and taxonomy of Cryptosphaeria species occurring in the western United States on the basis of morphological characters and multilocus phylogenetic analyses of the ribosomal internal transcribed spacer region, parts of a ß-tubulin gene, the DNA-dependent RNA polymerase II second-largest subunit gene and the nuclear ribosomal large subunit gene. Cryptosphaeria multicontinentalis sp. nov is described from the Sierra Nevada and central coast of California on Populus tremuloides, P. balsamifera subsp. trichocarpa and P. fremontii. Cryptosphaeria pullmanensis is reported from a wide geographic area in the western United States on the main host, P. fremontii. The pathogen C. lignyota is reported for the first time from the Sierra Nevada of California on P. tremuloides. The phylogenetic analyses showed that C. multicontinentalis is a sister species to C. lignyota. Both species were closely related to C. subcutanea and more distantly related to C. pullmanensis. Characteristics of both teleomorph and anamorph of the newly introduced species C. multicontinentalis are described and illustrated.

pH-Dependent accumulation of anticancer compound on mycelia in fermentation of marine fungus.

The real-time distribution of anticancer 1403C in fermentation broth of marine fungus Halorosellinia sp. was investigated. It was closely related with pH variations, which was, 1403C in the supernatant decreased while that in the mycelia increased with pH rising. There was only 0.5 % of the total 1403C left in the supernatant when pH reached 7.0. The scanning electron microscope then provided information that compounds precipitated on the mycelia when pH rose. Then, the pH-regulation experiments proved that 1403C mainly secreted extracellular and easily dissolved in acidic condition but precipitated and absorbed on the mycelia with the increase of broth pH. Thereby, a pH-regulation strategy was proposed and applied to accumulate 1403C on the mycelia before draw-off of fermentation broth. It significantly simplified purification process and is critical for 1403C preparation of industrial scale.

Nutrition and bioprocess development for efficient biosynthesis of an antitumor compound from marine-derived fungus.

An integrated nutrition and bioprocess strategy was developed for improving the biosynthesis of an antitumor compound, 1403C, by a marine-derived fungus, Halorosellinia sp. (no. 1403). First, statistical design strategies were synthetically applied to optimize the nutritional composition. The resulting 1403C production reached 2.07 g/l, which was 143.5 % higher than the original production. However, it only produced 0.44 g/l of 1403C in 5-l bioreactor fermentation. Thus, the operating parameters including culture pH, dissolved oxygen, agitation speed, impeller type and inoculum level were considered to improve the fermentation process, and an effective control strategy for 1403C production by Halorosellinia sp. submerged in a 5-l bioreactor was established. When inoculating 0.22 g/l dry biomass, controlling dissolved oxygen not lower than 30 % during the growth phase but ranging between 30 and 40 % during the stationary phase, using a double-layer six-flat-blade Rushton disc turbine agitated at 400 rpm, keeping short-term low pH and rapid-rising pH with glucose starvation, the highest 1403C production was finally obtained at 1.32 g/l, which was promoted by 200 % compared to before optimization. Fermentation scale-up was finally performed in a 500-l bioreactor, and 1403C production of 1.09 g/l was obtained.

Inhibitory effects of terpenoids from the fermented broth of the ascomycete Stilbohypoxylon elaeicola YMJ173 on nitric oxide production in RAW264.7 macrophages.

A series of six isopimarane-type diterpene glycosides, along with an eremophilane-type sesquiterpene, i.e., elaeicolasides A-C (1-3, resp.), 16-(α-D-mannopyranosyloxy)isopimar-7-en-19-oic acid (4), hymatoxin K (5), hymatoxin L (6), and elaeicolalactone (7), were isolated from the AcOEt extract of the fermented broth of Stilbohypoxylon elaeicola YMJ173. Among these, 1-3 and 7 are new compounds based on their spectroscopic data and sugar composition analysis. The effects of 1-7 on the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells were evaluated. All these compounds inhibited NO production, detected as nitrite in the culture medium, in activated macrophages without any cytotoxicity at a concentration of 100 µM. Among these compounds, 2 showed a significant activity with the average maximum inhibition (E(max)) and median inhibitory concentration (IC(50)) values of 93.3±0.5% and 79.3±0.4 µM, respectively.

Niche differentiation of two sympatric species of Microdochium colonizing the roots of common reed.

BACKGROUND: Fungal endophyte communities are often comprised of many species colonizing the same host. However, little is known about the causes of this diversity. On the one hand, the apparent coexistence of closely related species may be explained by the traditional niche differentiation hypothesis, which suggests that abiotic and/or biotic factors mediate partitioning. For endophytes, such factors are difficult to identify, and are therefore in most cases unknown. On the other hand, there is the neutral hypothesis, which suggests that stochastic factors may explain high species diversity. There is a need to investigate to what extent each of these hypotheses may apply to endophytes. RESULTS: The niche partitioning of two closely related fungal endophytes, Microdochium bolleyi and M. phragmitis, colonizing Phragmites australis, was investigated. The occurrences of each species were assessed using specific nested-PCR assays for 251 field samples of common reed from Lake Constance, Germany. These analyses revealed niche preferences for both fungi. From three niche factors assessed, i.e. host habitat, host organ and season, host habitat significantly differentiated the two species. M. bolleyi preferred dry habitats, whereas M. phragmitis prevailed in flooded habitats. In contrast, both species exhibited a significant preference for the same host organ, i.e. roots. Likewise the third factor, season, did not significantly distinguish the two species. Differences in carbon utilization and growth temperature could not conclusively explain the niches. The inclusion of three unrelated species of Ascomycota, which also colonize P. australis at the same locations, indicated spatio-temporal niche partitioning between all fungi. None of the species exhibited the same preferences for all three factors, i.e. host habitat, host organ, and time of the season. CONCLUSIONS: The fungal species colonizing common reed investigated in this study seem to exploit niche differences leading to a separation in space and time, which may allow for their coexistence on the same host. A purely neutral model is unlikely to explain the coexistence of closely related endophytes on common reed.

Chemical constituents of the fermented broth of the ascomycete Theissenia cinerea 89091602.

One new betaenone, theissenoic acid (1), together with three new acetogenins, theissenolactones A-C (2-4, resp.), were isolated from the fermented broth of Theissenia cinerea 89091602 isolated in Taiwan. The structures of 1-4 were elucidated by spectroscopic methods. Biological tests revealed that 3 and 4 exhibited moderate growth-inhibitory activities against A549 lung cancer cell line with GI(50) values of 14.9 and 47.9 µM, respectively.

Levels of specificity of Xylaria species associated with fungus-growing termites: a phylogenetic approach.

Fungus-growing termites live in obligate mutualistic symbiosis with species of the basidiomycete genus Termitomyces, which are cultivated on a substrate of dead plant material. When the termite colony dies, or when nest material is incubated without termites in the laboratory, fruiting bodies of the ascomycete genus Xylaria appear and rapidly cover the fungus garden. This raises the question whether certain Xylaria species are specialised in occupying termite nests or whether they are just occasional visitors. We tested Xylaria specificity at four levels: (1) fungus-growing termites, (2) termite genera, (3) termite species, and (4) colonies. In South Africa, 108 colonies of eight termite species from three termite genera were sampled for Xylaria. Xylaria was isolated from 69% of the sampled nests and from 57% of the incubated fungus comb samples, confirming high prevalence. Phylogenetic analysis of the ITS region revealed 16 operational taxonomic units of Xylaria, indicating high levels of Xylaria species richness. Not much of this variation was explained by termite genus, species, or colony; thus, at level 2-4 the specificity is low. Analysis of the large subunit rDNA region, showed that all termite-associated Xylaria belong to a single clade, together with only three of the 26 non-termite-associated strains. Termite-associated Xylaria thus show specificity for fungus-growing termites (level 1). We did not find evidence for geographic or temporal structuring in these Xylaria phylogenies. Based on our results, we conclude that termite-associated Xylaria are specific for fungus-growing termites, without having specificity for lower taxonomic levels.

Weed Roots Facilitate the Spread of Rosellinia necatrix, the Causal Agent of White Root Rot.

Rosellinia necatrix causes white root rot in various plants, including the Japanese pear. PCR assays using specific primers for R. necatrix detected the fungus on the roots of nine weed species from infested pear orchards. The soil inoculation experiment revealed that the spread of R. necatrix was similar between weed-mowed and non-weed-mowed treatments under field conditions. The spread of R. necatrix was also observed when rescue grass (Bromus catharticus) was grown in planter boxes under greenhouse conditions, but was limited without the grass, suggesting that some weeds facilitate the spread of R. necatrix in soil.

Effect of a fungus, Hypoxylon spp., on endophytes in the roots of Asparagus.

The fungal isolate Hypoxylon spp. (Sj18) was isolated from the root of pecan. It might have effects on the plant's stress tolerance and endophytic community. Inoculation experiments were carried out on the roots of Asparagus with normal and inactivated Sj18, and the diversity and community structure of endophytes in the root of inoculated Asparagus were studied. It was found that Sj18 fungi affected the endophytic community of Asparagus roots. From being a low-abundance genus, the salt-tolerant bacterium Halomonas became the dominant genus. In order to verify that Sj18 can improve salt tolerance, Arabidopsis thaliana was inoculated with Sj18 in a salt tolerance test. The result showed that A. thaliana grew better in a high salt environment after inoculation with Sj18. Sj18 changed the microbe diversity, community composition and structure of endophytes in the roots of Asparagus, which increased the bacterial diversity. A total of 16 phyla and 184 genera of bacteria were detected. However, the diversity of fungi decreased.

New ß-Lactone with Tea Pathogenic Fungus Inhibitory Effect from Marine-Derived Fungus MCCC3A00957.

Fusarium solani H915 (MCCC3A00957), a fungus originating from mangrove sediment, showed potent inhibitory activity against tea pathogenic fungus Pestalotiopsis theae. Successive chromatographic separation on an ethyl acetate (EtOAc) extract of F. solani H915 resulted in the isolation of five new alkenoic diacid derivatives: fusarilactones A-C (1-3), and fusaridioic acids B (4) and C (5), in addition to seven known compounds (6-12). The chemical structures of these metabolites were elucidated on the basis of UV, IR, HR-ESI-MS, and NMR spectroscopic data. The antifungal activity of the isolated compounds was evaluated. Compounds with a ß-lactone ring (1, 2, and 7) exhibited potent inhibitory activities, while none of the other compounds show activity. The ED50 values of the compounds 1, 2, and 7 were 38.14 ± 1.67, 42.26 ± 1.96, and 18.35 ± 1.27 µg/mL, respectively. In addition, inhibitory activity of these compounds against 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene expression was also detected using real-time RT-PCR. Results indicated that compounds 1, 2, and 7 may inhibit the growth of P. theae by interfering with the biosynthesis of ergosterol by down-regulating the expression of HMG-CoA synthase.