Potential role for saccharopine reductase in swainsonine metabolism in endophytic fungus, Undifilum oxytropis

Locoweed plants in the southwestern United States often harbour a slow-growing endophytic fungus, Undifilum oxytropis (Phylum: Ascomycota; Order: Pleosporales), which produces a toxic alkaloid, swainsonine. Consumption of U. oxytropis by grazing animals induces a neurological disorder called locoism for which the toxic alkaloid swainsonine has been reported to be the causal agent. Little is known about the biosynthetic pathway of swainsonine in endophytic fungi, but previous studies on non-endophytic ascomycetous fungi indicate that pipecolic acid and saccharopine are key intermediates. We have used degenerate primers, Rapid amplification of cDNA ends (RACE)-PCR and inverse PCR to identify the gene sequence of U. oxytropis saccharopine reductase. To investigate the role of this gene product in swainsonine metabolism, we have developed a gene deletion system for this slow-growing endophyte based on our recently established transformation protocol. A strain of U. oxytropis lacking saccharopine reductase had decreased levels of saccharopine and lysine along with increased accumulation of pipecolic acid and swainsonine. Thus, saccharopine reductase influences the accumulation of swainsonine and its precursor, pipecolic acid, in U. oxytropis.     

An Endophytic Sanguinarine-Producing Fungus from Macleaya cordata,Fusarium proliferatum BLH51

Fermentation processes using sanguinarine-producing fungi other than Macleaya cordata may be an alternative way to produce sanguinarine (SA), which is a quaternary benzo[c]phenanthridine alkaloid possessing antibacterial, anthelmintic, and anti-inflammatory properties. In this study, a SA-producing endophytic fungus strain BLH51 was isolated from the leaves of M. cordata grown in the Dabie Mountain, China. Strain BLH51 produced SA when grown in potato dextrose liquid medium. The amount of SA produced by this endophytic fungus was quantified to be 178 μg/L by HPLC, substantially lower than that produced by the host tissue. The fungal SA—which was analyzed by thin layer chromatography and high-performance liquid chromatography—was shown to be identical to authentic SA. Strain BLH51 was identified as Fusarium proliferatum based on the morphological characteristics and nuclear ribosomal DNA ITS sequence analysis. To the best of our knowledge, this is the first report concerning the isolation and identification of endophytic SA-producing fungi from the host plant, which further proved that endophytic fungi are valuable reservoirs of bioactive compounds.     

Efficient strategy for maintaining and enhancing the huperzine A production of Shiraia sp. Slf14 through inducer elicitation

Huperzine A (HupA), a naturally occurring lycopodium alkaloid, is a potent, highly specific and reversible inhibitor of acetylcholinesterase and is a potential treatment for Alzheimer’s disease. However, isolating HupA from Huperziaceae plants is inefficient; thus, extracting this compound from endophytic fungi may be more controllable and sustainable. However, the large-scale production of this chemical from endophytes is limited by the innate instability of endophytic fungi. In this study, we maintained the stability and viability of the HupA-producing endophytic fungus Shiraia sp. Slf14 and enhanced the HupA titers during fermentation by adding Huperzia serrata extracts (HSE), l-lysine, and acetic acid into the culture as inducers. Adding trace amounts of HupA clearly improved the HupA production of Shiraia sp. Slf14, reaching a maximum content of approximately 40 μg g^?1. Moreover, the addition of HSE and l-lysine promoted HupA production in the flask fermentation. The aforementioned bioprocessing strategy may be potentially applied to other endophytic fungal culture systems for the efficient production of plant secondary metabolites.     

Genomic and transcriptomic analysis of the endophytic fungus Pestalotiopsis fici reveals its lifestyle and high potential for synthesis of natural products

Background

In recent years, the genus Pestalotiopsis is receiving increasing attention, not only because of its economic impact as a plant pathogen but also as a commonly isolated endophyte which is an important source of bioactive natural products. Pestalotiopsis fici Steyaert W106-1/CGMCC3.15140 as an endophyte of tea produces numerous novel secondary metabolites, including chloropupukeananin, a derivative of chlorinated pupukeanane that is first discovered in fungi. Some of them might be important as the drug leads for future pharmaceutics.

Results

Here, we report the genome sequence of the endophytic fungus of tea Pestalotiopsis fici W106-1/CGMCC3.15140. The abundant carbohydrate-active enzymes especially significantly expanding pectinases allow the fungus to utilize the limited intercellular nutrients within the host plants, suggesting adaptation of the fungus to endophytic lifestyle. The P. fici genome encodes a rich set of secondary metabolite synthesis genes, including 27 polyketide synthases (PKSs), 12 non-ribosomal peptide synthases (NRPSs), five dimethylallyl tryptophan synthases, four putative PKS-like enzymes, 15 putative NRPS-like enzymes, 15 terpenoid synthases, seven terpenoid cyclases, seven fatty-acid synthases, and five hybrids of PKS-NRPS. The majority of these core enzymes distributed into 74 secondary metabolite clusters. The putative Diels-Alderase genes have undergone expansion.

Conclusion

The significant expansion of pectinase encoding genes provides essential insight in the life strategy of endophytes, and richness of gene clusters for secondary metabolites reveals high potential of natural products of endophytic fungi.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-014-1190-9) contains supplementary material, which is available to authorized users.     

云南会泽铅锌矿废弃矿渣堆常见植物内生真菌多样性

从云南会泽铅锌矿废弃矿渣堆上的常见植物硬毛南芥(Arabis hirsuta)、毛萼香茶菜(Rabbosia eriocalyx)和倒挂刺(Rosa longicuspis)等6种植物的690个组织块中共分离得到内生真菌495株,内生真菌的分离频率在0.42—0.93之间,平均为0.72,所有植物茎内生真菌的分离频率都明显高于叶(P<0.05)。经形态学鉴定,内生真菌分属于茎点霉属(Phoma)、交链孢属(Alternaria)和派伦霉属(Peyronellaea)等20个分类单元,其中茎点霉属和派伦霉属为该废弃矿渣堆上常见植物的优势内生真菌属。6种植物内生真菌的多样性指数在1.05—2.29之间,与其它非重金属污染环境植物内生真菌的多样性指数相似,说明在重金属污染地区仍然存在多种重金属耐受的内生真菌种类。6种植物内生真菌的相似性系数(0.455—0.833)表明,会泽铅锌矿区植物内生真菌的宿主专一性较小。     

Production of a benzylated flavonoid from 5,7,3′,4′,5′-pentamethoxyflavanone by Penicillium griseoroseum

Penicillium griseoroseum, isolated as an endophytic microorganism from Coffeea arabica seeds, was grown in Czapeck's medium containing 5,7,3′,4′,5′-pentamethoxyflavanone. The fungus incorporated a dimethylated tetraketide, clavatol, a typical fungal secondary metabolite, into the flavanone structure at C-6, resulting in a novel benzylated flavonoid. Clavatol was also found free in the fungus extract. The compounds were isolated by chromatographic procedures and identified by extensive spectroscopic studies, including MS/MS and 1D and 2D NMR. The process probably involves enzymes catalyzing C–C bound formation, which is uncommon in fungi. The possibility of fungi participation in the biosynthesis of plant benzylated flavonoids is discussed.     

Antiproliferative,Antifungal, and Antibacterial Activities of Endophytic Alternaria Species from Cupressaceae

Recent research has shown the bioprospecting of endophytic fungi from Cupressaceae. Here, we further uncover that the healthy cypress plants such as Cupressus arizonica, Cupressus sempervirens var. cereiformis, and Thuja orientalis host highly bioactive endophytic Alternaria fungal species. Indeed, endophytic Alternaria alternata, Alternaria pellucida, and Alternaria tangelonis were recovered from healthy Cupressaceous trees. Biodiversity and bioactivity of recovered endophytic Alternaria species were a matter of biogeography and host identity. We further extracted such Alternaria’s metabolites and highlighted their significant antiproliferative, growth inhibitory, and antibacterial activities against the model target fungus Pyricularia oryzae and the model pathogenic bacteria Bacillus sp., Erwinia amylovora, and Pseudomonas syringae. In vitro assays also indicated that endophytic Alternaria species significantly inhibited the growth of cypress fungal phytopathogens Diplodia seriata, Phaeobotryon cupressi, and Spencermartinsia viticola. In conclusion, since the recovered Alternaria species were originally reported as pathogenic and allergenic fungi, our findings suggest a possible ecological niche for them inside the foliar tissues of Cupressaceous trees. Moreover, in this study, the significant bioactivities of endophytic Alternaria species in association with Cupressaceae plant family are reported.     

Biodiversity of endophytic fungi in different leaf ages of Calotropis procera and their antimicrobial activity

Calotropis procera has many important medicinal properties with proven pharmacological potential. Some of these properties may be mediated by its fungal endophytes. This study analyzed, for the first time, the community of endophytic fungi of C. procera outside its region of origin. A total of 156 fungal isolates distributed across 19 taxa were obtained from 468 fragments of C. procera leaves at different stages of maturation. The rate of endophyte colonization increased with the leaf age/development. The dominant species of endophytic fungi of C. procera introduced in Northeast Brazil were different from those found in studies on the same species and other species of the same genus in native regions. The dominant endophyte was Phaeoramularia calotropidis (63.5 %), followed by Guignardia bidwellii (21.1 %). Six isolates of endophytic fungi showed antimicrobial activity against human pathogenic micro-organisms and one plant pathogenic fungus. The antibacterial activity was more intense than the antifungal activity. The endophytic Curvularia pallescens (URM 6048) stood out inhibited Gram-positive bacteria, Staphylococcus aureus, Streptococcus pyogenes, the plant pathogenic fungus Colletotrichum dematium. Ecological and biotechnological aspects of endophytic mycota are discussed.     

Diversity,regulation, and evolution of the gibberellin biosynthetic pathway in fungi compared to plants and bacteria

Bioactive gibberellins (GAs) are diterpene plant hormones that are biosynthesized through complex pathways and control diverse aspects of growth and development. GAs were first isolated as metabolites of a fungal rice pathogen, Gibberella fujikuroi, since renamed Fusarium fujikuroi. Although higher plants and the fungus produce structurally identical GAs, significant differences in their GA pathways, enzymes involved and gene regulation became apparent with the identification of GA biosynthetic genes in Arabidopsis thaliana and F. fujikuroi. Recent identifications of GA biosynthetic gene clusters in two other fungi, Phaeosphaeria spp. and Sphaceloma manihoticola, and the high conservation of GA cluster organization in these distantly related fungal species indicate that fungi evolved GA and other diterpene biosynthetic pathways independently from plants. Furthermore, the occurrence of GAs and recent identification of the first GA biosynthetic genes in the bacterium Bradyrhizobium japonicum make it possible to study evolution of GA pathways in general.In this review, we summarize our current understanding of the GA biosynthesis pathway, specifically the genes and enzymes involved as well as gene regulation and localization in the genomes of different fungi and compare it with that in higher and lower plants and bacteria.     

Molecular characterization of a seed transmitted clavicipitaceous fungus occurring on dicotyledoneous plants (Convolvulaceae)

Ergoline alkaloids (syn. ergot alkaloids) are constituents of clavicipitaceous fungi (Ascomycota) and of one particular dicotyledonous plant family, the Convolvulaceae. While the biology of fungal ergoline alkaloids is rather well understood, the evolutionary and biosynthetic origin of ergoline alkaloids within the family Convolvulaceae is unknown. To investigate the possible origin of ergoline alkaloids from a plant-associated fungus, 12 endophytic fungi and one epibiotic fungus were isolated from an ergoline alkaloid-containing Convolvulaceae plant, Ipomoea asarifolia Roem. & Schult. Phylogenetic trees constructed from 18S rDNA genes as well as internal transcribed spacer (ITS) revealed that the epibiotic fungus belongs to the family Clavicipitaceae (Ascomycota) whereas none of the endophytic fungi does. In vitro and in vivo cultivation on intact plants gave no evidence that the endophytic fungi are responsible for the accumulation of ergoline alkaloids in I. asarifolia whereas the epibiotic clavicipitaceous fungus very likely is equipped with the genetic material to synthesize these compounds. This fungus resisted in vitro and in vivo cultivation and is seed transmitted. Several observations strongly indicate that this plant-associated fungus and its hitherto unidentified relatives occurring on different Convolvulaceae plants are responsible for the isolated occurrence of ergoline alkaloids in Convolvulaceae. This is the first report of an ergot alkaloid producing clavicipitaceous fungus associated with a dicotyledonous plant.Data deposition: The sequences reported in this paper have been deposited in the GenBank (accession numbers are given in the text and in Fig. 3) Dedicated to Dr. Dr. h. c. mult. Albert Hofmann, the great pioneer of ergot research, on the occasion of his 100th birthday     

Paeonol produced by Chaetomium sp., an endophytic fungus isolated from Paeonia suffruticosa

In order to research the relationship between endophytic fungus and active ingredients in medicinal Paeonia suffruticosa, a total of 57 fungal isolates were isolated from the roots, stems, leaves and buds of medicinal plant Paeonia ostii; mycelium was collected after these fungal isolates were fermented on PDA medium for a few days; then the mycelium products were extracted; their extracts were analyzed by gas chromatography–mass spectrometry. With this method, a strain endophytic fungi named J1-2 which can produce paeonol was screened. Paeonol produced by J1-2 was analyzed by using a high resolution mass spectrometer (HRMS) and nuclear magnetic resonance (NMR). The potential paeonol-procucing named J1-2 was identified Chaetomium based on morphological characteristics and ITS sequence analysis. The current research initially indicates that endophytic fungi can affect the potency of peony. At the same time it also indicates that the numerous endophytic fungi inside the medicinal Paeonia suffruticosa are precious resource for the pharmaceutical natural products that are originally from the Paeonia suffruticosa.     

Isolation of an endophytic fungus producing baccatin III from Taxus wallichiana var. mairei

The objective of this study was to isolate endophytic fungi producing baccatin III from yew for the purpose of baccatin III and paclitaxel manufacture. Surface sterilized bark of Taxus wallichiana var. mairei was used as source material with potato dextrose agar culture medium for isolation of endophytic fungi. Fungal cultures were extracted with a mixture of chloroform/methanol (1:1, v/v) and the baccatin III in the extracts was determined and authenticated with LC–MS. An endophytic fungus that produced baccatin III was identified by ITS rDNA and 26S D1/D2 rDNA sequencing. A total of 192 endophytic fungal strains were isolated from T. wallichiana var. mairei. Only one of the 192 strains produced baccatin III and it was identified as Diaporthe phaseolorum. The productivity of this strain cultured in PDA culture medium was 0.219 mg/l. The isolated endophytic fungus produced baccatin III at a relatively high level and shows promise as a producing strain for baccatin III and paclitaxel manufacture after strain improvement.     

Antifungal Metabolites Produced by Chaetomium globosum No.04, an Endophytic Fungus Isolated from Ginkgo biloba

The fungal endophyte Chaetomium globosum No.04 was isolated from the medicinal plant Ginkgo biloba. The crude extract of the fungus fermentation were active in the agar-diffusion tests against the phytopathogenic fungi Rhizopus stolonifer and Coniothyrium diplodiella. Further bioassay-guided chemical investigation led to the isolation and purification of six alkaloids and three non-targeted compounds from 50 L fermentation of this endophytic fungus and their structures were elucidated as chaetoglobosin A, C, D, E, G, R (1-6), ergosterol, allantoin and uracil, by means of spectroscopic analysis. Compounds 1-6 showed significant growth inhibitory activity against R. stolonifer and C. diplodiella at a concentration of 20 μg/disc. We present here, for the first time, the potent antifungal activity of chaetoglobosins from endophytic fungi against two important phytopathogenic fungi R. stolonifer and C. diplodiella.     

Development of methods for detection and Agrobacterium-mediated transformation of the sterile,endophytic fungus Muscodor albus

Symbiotic endophytes, unlike plant pathogens, do not usually induce visible host response. This may constraint the researcher's decision whether a plant has been successfully infected by the endophyte. In order to properly study the establishment, development and progress of an endophyte in the host plant and host-endophyte interactions, methods for the identification and localization of endophytic microorganisms are needed. Towards this aim, we focused at two levels: (A) We constructed M. albus-specific primers for polymerase chain reaction (PCR). In vitro, these primers specifically detected only M. albus strains and not isolates of related fungi (such as Daldinia sp. and a Xylariaceae sp.). (B) For direct visualization of the fungi, we inserted a reporter gene (gfp) into M. albus hyphae using Agrobacterium-mediated transformation. Since M. albus is a sterile fungus (i.e., without spores or fungal fruiting bodies), we used chopped fungal mycelium for the transformation procedure. We transformed three different isolates of M. albus using Agrobacterium-mediated transformation. Fifty-nine different transformants were collected with a transformation efficacy of 0.0004–0.0026%. Although PCR-based detection and direct visualization of the transformants in planta were unsuccessful, all tested transformants (with one exception) exhibited similar biological activity to their cognate wild type. This work provides a significant step forward in molecular research of the relationships between this endophytic genus and their hosts.     

Specificity in the interaction between an epibiotic clavicipitalean fungus and its convolvulaceous host in a fungus/plant symbiotum

Ipomoea asarifolia and Turbina corymbosa (Convolvulaceae) are associated with epibiotic clavicipitalean fungi responsible for the presence of ergoline alkaloids in these plants. Experimentally generated plants devoid of these fungi were inoculated with different epibiotic and endophytic fungi resulting in a necrotic or commensal situation. A symbiotum of host plant and its respective fungus was best established by integration of the fungus into the morphological differentiation of the host plant. This led us to suppose that secretory glands on the leaf surface of the host plant may play an essential role in ergoline alkaloid biosynthesis which takes place in the epibiotic fungus.Key words: ergoline alkaloids, ipomoea, turbina, convolvulaceae, claviceps, balansia, clavicipitaceae, penicillium, plant-fungus symbiotum     

Formation of arthroconidia during regeneration and selection of transformed Epichloë festucae protoplasts

Transformation is an essential tool for modern fungal research and has played a fundamental role in gaining insight into gene function. Polyethylene glycol (PEG)-mediated transformation of protoplasts is the most commonly used method for genetic transformation of filamentous fungi. Selectable marker genes, that confer resistance to antibiotics, are generally incorporated with the DNA of interest, allowing transformed cells to grow through the antibiotic overlay. Colonies arising from transformed fungal cells are sub-cultured and further analysed. However, the morphological state of the fungal cells during the transformation procedure has been largely overlooked. We investigated the morphological appearance of transformed fungal cells prior to their emergence through the antibiotic overlay. Hyphae appeared to segment and bulge, reminiscent of arthroconidia, an asexual spore typically produced by segmentation of pre-existing hyphae. Selective expression of eGFP under the control of a spore specific promoter, PcatA, in these cells confirmed their spore-like nature. Reducing the oxygen availability to surface-grown cultures partially recapitulated this morphological form. A GFP fusion to the cell wall integrity MAP kinase MpkA localised to the arthroconidia nuclei suggesting the cell wall integrity signalling pathway modulates cell wall stress responses in arthroconidia. This dramatic morphological change was also observed in transformed Magnaporthe oryzae cells suggesting it may be a more general phenomenon in filamentous fungi. Given the changes in cellular structure and spore-like appearance, these observations may have technical implications for deleting genes involved in these processes in Epichloë festucae and, more broadly, a range of fungal species.     

Endophyte communities vary in the needles of Norway spruce clones

Endophytic fungi show no symptoms of their presence but can influence the performance and vitality of host trees. The potential use of endophytes to indicate vitality has been previously realized, but a standard protocol has yet to be developed due to an incomplete understanding of the factors that regulate endophyte communities. Using a culture-free molecular approach, we examined the extent to which host genotype influences the abundance, species richness, and community composition of endophytic fungi in Norway spruce needles. Briefly, total DNA was extracted from the surface-sterilized needles of 30 clones grown in a nursery field and the copy number of the fungal internal transcribed spacer (ITS) region of ribosomal DNA was estimated by quantitative PCR. Fungal species richness and community composition were determined by denaturing gradient gel electrophoresis and DNA sequencing. We found that community structure and ITS copy number varied among spruce clones, whereas species richness did not. Host traits interacting with endophyte communities included needle surface area and the location of cuttings in the experimental area. Although Lophodermium piceae is considered the dominant needle endophyte of Norway spruce, we detected this species in only 33 % of samples. The most frequently observed fungus (66 %) was the potentially pathogenic Phoma herbarum. Interestingly, ITS copy number of endophytic fungi correlated negatively with the richness of ectomycorrhizal fungi and thus potential interactions between fungal communities and their influence on the host tree are discussed. Our results suggest that in addition to environmental factors, endophyte communities of spruce needles are determined by host tree identity and needle surface area.     

Colonization of a Submersed Aquatic Plant, Eurasian Water Milfoil (Myriophyllum spicatum), by Fungi under Controlled Conditions

A laboratory assay to assess colonization of a submersed aquatic plant, Eurasian water milfoil (Myriophyllum spicatum), by fungi was developed and used to evaluate the colonization potential of Colletotrichum gloeosporioides, Acremonium curvulum, Cladosporium herbarum, Aureobasidium pullulans, a Paecilomyces sp., and an unidentified sterile, septate fungus. Stem segments of plants were first immersed in suspensions of fungal propagules for 24 h and then washed to remove all but the tightly attached component of the population. Inoculation was followed by two growth cycles of 3 days each. At the start of each cycle, washed plants were transferred to a mineral salts medium to provide an opportunity for the attached fungal populations to grow. After each growth period, plants were again washed, and fungal populations in the medium (nonattached), loosely attached and tightly attached to the plant, and within the plant (endophytic) were assayed by dilution plating. The fungi differed in the extent to which they attached to water milfoil and in their ability to grow in association with it. There were relatively few significant differences among the tightly attached fungal populations after 24 h, but growth of the better colonizers led to a greater number of significant differences after 4 and 7 days. In addition, the better colonizers showed sustained regrowth of loosely and nonattached fungal propagules in the face of intermittent removal by washing. A milfoil pathogen, C. gloeosporioides, was the only endophytic colonizer; it was also among the best epiphytic colonizers but was not demonstrably better than A. curvulum, a fungus commonly found as an epiphyte on watermilfoil. The yeastlike hyphomycete Aureobasidium pullulans was the only fungus that consistently failed to establish an increasing population on the plant.     

Botryorhodines A-D, antifungal and cytotoxic depsidones from Botryosphaeria rhodina, an endophyte of the medicinal plant Bidens pilosa

An endophytic fungus (Botryosphaeria rhodina) was isolated from the stems of the medicinal plant Bidens pilosa (Asteraceae) that is known for its anti-inflammatory, antiseptic and antifungal effects. The ethyl acetate extract of the fungal isolate exhibits significant antifungal activity as well as potent cytotoxic and antiproliferative effects against several cancer cell lines. Activity-guided fractionation resulted in the isolation of a complex of four depsidones, botryorhodines A-D and the auxin indole carboxylic acid. Botryorhodine A and B show moderate to weak cytotoxic activities against HeLa cell lines with a CC₅₀ of 96.97 μM and 36.41 μM, respectively. In addition, they also show antifungal activity against a range of pathogenic fungi such as Aspergillus terreus (MIC 26.03 μM for botryorhodine A and 49.70 μM for B) and the plant pathogen Fusarium oxysporum (MIC 191.60 μM for botryorhodine A and 238.80 μM for B). A potential role of the endophyte in modulating fungal populations living within or attacking the host plant is discussed.