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.

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.

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.

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.

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.

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.

A MYST Histone Acetyltransferase Modulates Conidia Development and Secondary Metabolism in Pestalotiopsis microspora, a Taxol Producer.

Reverse genetics is a promising strategy for elucidating the regulatory mechanisms involved in secondary metabolism and development in fungi. Previous studies have demonstrated the key role of histone acetyltransferases in transcriptional regulation. Here, we identified a MYST family histone acetyltransferase encoding gene, mst2, in the filamentous fungus Pestalotiopsis microspora NK17 and revealed its role in development and secondary metabolism. The gene mst2 showed temporal expression that corresponded to the conidiation process in the wild-type strain. Deletion of mst2 resulted in serious growth retardation and impaired conidial development, e.g., a delay and reduced capacity of conidiation and aberrant conidia. Overexpression of mst2 triggered earlier conidiation and higher conidial production. Additionally, deletion of mst2 led to abnormal germination of the conidia and caused cell wall defects. Most significantly, by HPLC profiling, we found that loss of mst2 diminished the production of secondary metabolites in the fungus. Our data suggest that mst2 may function as a general mediator in growth, secondary metabolism and morphological development.

Roles of phospholipid methyltransferases in pycnidia development, stress tolerance and secondary metabolism in the taxol-producing fungus Pestalotiopsis microspore.

Phosphatidylcholine (PC) is an important membrane component of the eukaryotic cell. In yeast fungi, two phospholipid methyltransferases catalyze consecutive steps of methylation in the formation of phosphatidylcholine from phosphatidylethanolamine. However, roles of phospholipid methyltransferases in filamentous fungi remains less investigated. We report here the characterization of two genes, choA and choC, that putatively encoded phospholipid methyltransferases in the taxol-producing fungus Pestalotiopsis microspora. Deletion of choC resulted in defects in PC production, vegetative growth and development of asexual structure. The mutant strains exhibited multiple morphological abnormalities, e.g. swollen hyphal tips and enhanced hyphal branching, and even mycelial autolysis. Some novel roles for the genes were also revealed, for instance, the deletion of either choC or choA impaired the development of pycnidia and conidia, the cell wall integrity. The mutant strains displayed a hypersensitivity to stress conditions, e.g. osmotic stress, cold and metal ions. The osmotic hypersensitivity indicates a crosstalk of PC pathways to other signaling pathways, such as the HOG pathway. Still more, choA, but not choC, was required for the production of secondary metabolites, e.g. pestalotiollide B, suggesting distinct roles of the two genes. This work would contribute to better understanding the function of phospholipid methyltransferases in fungi.

Induced effect of Ca2+ on dalesconols A and B biosynthesis in the culture of Daldinia eschscholzii via calcium/calmodulin signaling.

Dalesconols (dalesconols A and B) were isolated from Daldinia eschscholzii and have remarkable immunosuppressive activity. In this study, the response of fungal growth, intra- and extracellular Ca2+, and dalesconols production after CaCl2 addition were reported for the first time. After supplementation with 5 mM Ca2+ at 24 h, dalesconols production reached 84.33 mg/L, which resulted in a 1.57-fold enhancement compared to the control. The key role of calcium/calmodulin signaling in dalesconols biosynthesis was confirmed by treatment with Ca2+ channel and calmodulin inhibitors. The transcriptional levels of dalesconols biosynthetic genes were up-regulated after CaCl2 addition and down-regulated after inhibitors were added. The results demonstrated that Ca2+ addition induces dalesconols biosynthesis through up-regulation of dalesconols biosynthesis genes via regulation of calcium/calmodulin signaling. This study provided an efficient strategy for improving dalesconols production and would facilitate further research on the biosynthesis and regulation of dalesconols.

COP9 signalosome subunit PfCsnE regulates secondary metabolism and conidial formation in Pestalotiopsis fici.

The COP9 signalosome (CSN) is a highly conserved multiprotein complex in all eukaryotes and involved in regulation of organism development. In filamentous fungi, several lines of evidence indicate that fungal development and secondary metabolism (SM) are mediated by the fifth subunit of CSN, called CsnE. Here we uncover a connection with CsnE and conidial formation as well as SM regulation in the plant endophytic fungus Pestalotiopsis fici. A homology search of the P. fici genome with CsnE, involved in sexual development and SM in Aspergillus nidulans, identified PfCsnE. Deletion of PfcsnE resulted in a mutant that stopped conidial production, but the conidia are recovered in a PfcsnE complemented strain. This indicates that PfCsnE is required for the formation of conidia. Secondary metabolite analysis demonstrated that the ΔPfcsnE strain produced more chloroisosulochrin, less ficiolide A production in comparison to wild type (WT). Transcriptome analysis of WT and ΔPfcsnE strains indicated that PfcsnE impacts the expression levels of 8.37% of 14,797 annotated genes. Specifically, nine biosynthetic gene clusters (BGCs) were up-regulated and three BGCs were down-regulated by PfCsnE. Our results suggest that PfCsnE plays major roles in SM regulation and conidial development in P. fici.

Production, Purification and Characterisation of a Potential Fibrinolytic Protease from Endophytic Xylaria curta by Solid Substrate Fermentation.

The present investigation highlights the optimal conditions for production of a non-toxic, bi-functional fibrinolytic enzyme xylarinase produced by endophytic fungus Xylaria curta by solid substrate fermentation using rice chaff medium. The purified enzyme is a monomeric protein with a molecular mass of ∼33 kDa. The enzyme exhibits cleavage of Aα and Bß chains of fibrin(ogen) and has no effect on γ chain. The optimal fibrinolytic activity of the enzyme was observed at 35 °C and pH 8. The fibrinolytic activity was enhanced in the presence of Ca2+, whereas it was completely inhibited in the presence of Fe2+ and Zn2+ ions and inhibitors like EDTA and EGTA suggesting it to be a metalloprotease. The K m and V max of the enzyme for azocasein were 326 µM and 0.13 µM min-1. The N-terminal sequence of the enzyme (SNGPLPGGVVWAG) was same when compared to xylarinase isolated from culture broth of X. curta. Thus, xylarinase could be exploited as a potent clot busting enzyme which could be produced on large scale using solid substrate fermentation.

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.

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.

Enhancement of dalesconols A and B production via upregulation of laccase activity by medium optimization and inducer supplementation in submerged fermentation of Daldinia eschscholzii.

Dalesconols (dalesconols A and B) are novel polyketides with strong immunosuppressive activity produced by Daldinia eschscholzii. In this work, the effects of different media (M1, M2, and M3) on fungus growth and dalesconols biosynthesis were firstly tested and compared. Intermediates and enzyme analysis indicated that laccase had the major contribution to dalesconols biosynthesis. The key role of laccase on dalesconols biosynthesis was further experimentally confirmed, which suggested that the modified M2 was more favored for laccase and dalesconols production. Thereafter, the medium composition was optimized by RSM with a fermentation titer of 36.66 mg/L obtained. Furthermore, Ca(2+) induction was employed to up-regulate of laccase activity and further enhanced dalesconols production (76.90 mg/L), which was 308% higher than that in M2. In addition, dalesconols production reached 63.42 mg/L in scale-up experiments. This work indicated great potential of laccase as a key enzyme on regulation of dalesconols production.

Identification and characterization of Pestalotiopsis-like fungi related to grapevine diseases in China.

Pestalotiopsis-like fungi are an important plant pathogenic genus causing postharvest fruit rot and trunk diseases in grapevine in many countries. Pestalotiopsis-like fungi diseases were studied in vineyards in nine provinces across China. Multi-gene (ITS, ß-tubulin and tef1) analysis coupled with morphology showed that a Neopestalotiopsis sp. and Pestalotiopsis trachicarpicola are associated in causing grapevine fruit rot and trunk diseases in China. Pestalotiopsis trachicarpicola is reported as the causative agent of grapevine diseases in the world for the first time. Neopestalotiopsis sp. caused significantly longer lesions than the other taxon present. This study represents the first attempt to identify and characterize the Pestalotiopsis-like fungi causing grapevine diseases in China using both morphological and molecular approaches.

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.

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.

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.

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.