Endophytic Filamentous Fungi Research Articles

BIODIVERSITY AND ANTIBACTERIAL ACTIVITIES OF ENDOPHYTIC FUNGI ASSOCIATED WITH UNCARIA GAMBIER (HUNTER) ROXB. VAR. UDANG FROM JASINGA, BOGOR, INDONESIA

Endophytic fungi living in medicinal plant tissues are potential bioactive metabolite sources for drug discovery and development. However, the research regarding endophytic fungi associated with Indonesian medicinal plants is still limited. This study aimed to explore the biodiversity of endophytic fungi associated with the medicinal plant Uncaria gambir (Hunter) Roxb. var. udang and their antibacterial activities. Twenty-seven endophytic filamentous fungi were isolated from the surface-sterilized leaves, stems, and roots of Uncaria gambir (Hunter) Roxb. var. udang collected in Jasinga, Bogor, West Java, Indonesia. The observation based on the morphological characters both macroscopically and microscopically, as well as chemical profiling using thin layer chromatography (TLC), revealed that some of the isolated endophytic fungi were identical. Overall, the isolated endophytic fungi consist of six Coelomycetes, three Dematiaceae, four Pestalotiopsis sp., two Penicillium sp., one Aspergillus terreus, and two fungi were unidentified. Antibacterial activities of the ethyl acetate extract from the isolated endophytic fungi associated with Uncaria gambir (Hunter) Roxb. var. udang were observed against E. coli, B. subtilis, S. aureus, and M. luteus. Twelve types of isolated endophytic fungi from Uncaria gambir (Hunter) Roxb. var. udang exhibited antibacterial activities.

Environmental differences between Japan and Indonesia provide endophyte diversity associated with Artemisia plant and variety of artemisinin derivatives in microbial conversion.

We compared the endophytic compositions of Artemisia plant from different environments (Japan and Indonesia) to demonstrate that the endophytic filamentous fungi in both species differed based on their environments. To prove that the species were identical, both Artemisia plants were identified by comparing the scanning electron micrographs of their pollens, as well as the nucleotide sequences (ribosomal internal transcribed spacer and mitochondrial maturase K) of the two gene regions. After isolating the endophytic filamentous fungi from each plant, we observed that those from Japan and Indonesia comprised 14 and 6 genera, respectively. We assumed that the genera, Arthrinium and Colletotrichum, which exist in both Artemisia species, were species-specific filamentous fungi, while the other genera were environment-dependent. In the microbial-conversion reaction with artemisinin as a substrate using Colletotrichum sp., the peroxy bridge of artemisinin, which is an active site for achieving antimalarial effect, was converted into an ether bond. However, the reaction using the environment-dependent endophyte did not eliminate the peroxy bridge. These endophytic reactions indicated the different roles of endophytes within Artemisia plants.

Red biocolorant from endophytic Talaromyces minnesotensis: production, properties, and potential applications.

Fungal colorants are gradually entering the global color market, given their advantages of being less harmful to human health, as well as having greater stability and biotechnological potential, compared to other natural sources. The present work concerns the isolation and identification of an endophytic filamentous fungus, together withthe chemical characterization and assessment of the fluorescence, toxicity, stability, and application potential of its synthesized red colorant. The endophytic fungus was isolated from Hymenaea courbaril, a tree from the Brazilian savannah, and was identified as Talaromyces minnesotensis by phenotypic and genotypic characterization. Submerged cultivation of the fungus resulted in the production of approximately 12 AU500 of a red biocolorant which according to LC-DAD-MS analysis is characterized by being a complex mixture of molecules of the azaphilone class. Regarding cytotoxicity assays, activity against human hepatoblastoma (HepG2) cells was only observed at concentrations above 5.0g L-1, while antimicrobial effects against pathogenic bacteria and yeast occurred at concentrations above 50.0g L-1. The biocolorant showed high stability at neutral pH values and low temperatures (10 to 20°C) and high half-life values (t1/2), which indicates potential versatility for application in different matrices, as observed in tests using detergent, gelatin, enamel, paint, and fabrics. The results demonstrated that the biocolorant synthesized by Talaromyces minnesotensis has potential for future biotechnological applications. KEY POINTS: • An endophytic fungus, which was isolated and identified, synthesize a red colorant. • The colorant showed fluorescence property, low toxicity, and application potential. • The red biocolorant was highly stable at pH 8.0 and temperatures below 20°C.

Phosphate Mobilization by Culturable Fungi and Their Capacity to Increase Soil P Availability and Promote Barley Growth.

Large-scale screening of 848 culturable soil and endophytic filamentous fungi and yeasts for the ability to mobilize inorganic and organic P compounds was performed. Five strains of filamentous fungi having the highest level of phosphate-mobilizing ability were selected: Penicillium bilaiae Pb14, P. bilaiae C11, P. rubens EF5, Talaromyces pinophilus T14, and Aspergillus sp. D1. These strains in vitro actively solubilized Ca, Al, and Fe phosphates and Ca phytate. The amount of mobilized P negatively correlated with pH of the medium and positively correlated with fungal biomass. The proposed mechanisms for P mobilization were acidification of the medium, organic acid release, and phosphatase activity. The fungi decreased pH of the medium from 7.0 to 2.3-5.0. Ten different organic acids were produced by fungi with pyruvic acid being a major component. Acid phosphatase activity varied from 0.12 EU to 0.84 EU, and alkaline phosphatase activity varied from 0.08 EU to 0.61 EU depending on the strain. Available P concentration in soil was increased by 13-28% after introduction of the fungi. The fungi also produced phytohormones auxins, salicylic acid, and abscisic acid. All the strains, except Aspergillus sp. D1, promoted elongation and increased biomass of barley seedlings grown in soil. Shoot P concentration increased by 17-26% after inoculation with P. bilaiae Pb14, T. pinophilus T14, and Aspergillus sp. D1. It was concluded that the selected fungal strains promoted plant growth due to P mobilization and phytohormone production.

In Silico Analysis of Functionalized Hydrocarbon Production Using Ehrlich Pathway and Fatty Acid Derivatives in an Endophytic Fungus.

Functionalized hydrocarbons have various ecological and industrial uses, from signaling molecules and antifungal/antibacterial agents to fuels and specialty chemicals. The potential to produce functionalized hydrocarbons using the cellulolytic, endophytic fungus, Ascocoryne sarcoides, was quantified using genome-enabled, stoichiometric modeling. In silico analysis identified available routes to produce these hydrocarbons, including both anabolic- and catabolic-associated strategies, and determined correlations between the type and size of the hydrocarbons and culturing conditions. The analysis quantified the limits of the wild-type metabolic network to produce functionalized hydrocarbons from cellulose-based substrates and identified metabolic engineering targets, including cellobiose phosphorylase (CP) and cytosolic pyruvate dehydrogenase complex (PDHcyt). CP and PDHcyt activity increased the theoretical production limits under anoxic conditions where less energy was extracted from the substrate. The incorporation of both engineering targets resulted in near-complete conservation of substrate electrons in functionalized hydrocarbons. The in silico framework was integrated with in vitro fungal batch growth experiments to support O2 limitation and functionalized hydrocarbon production predictions. The metabolic reconstruction of this endophytic filamentous fungus describes pathways for both specific and general production strategies of 161 functionalized hydrocarbons applicable to many eukaryotic hosts.

Antidiabetic xanthones with α-glucosidase inhibitory activities from an endophytic Penicillium canescens

Worldwide, 463 million people are affected by diabetes of which the majority is diagnosed with Type 2 Diabetes (T2D). T2D can ultimately lead to retinopathy, nephropathy, nerve damage, and amputation of the lower extremities. α-Glucosidase, responsible for converting starch to monosaccharides, is a key therapeutic target for the management of T2D. However, due to substantial side effects of currently marketed drugs, there is an urgent need for the discovery of new α-glucosidase inhibitors. In our ongoing efforts to identify novel α-glucosidase inhibitors from Nature, we are investigating the potential of endophytic filamentous fungi as sustainable sources of hits and/or leads for future antihyperglycemic drugs. Here we report one previously unreported xanthone (5) and two known xanthones (7 and 11) as α-glucosidase inhibitors, isolated from an endophytic Penicillium canescens, recovered from fruits of Juniperus polycarpos. The three xanthones 5, 7, and 11 showed inhibitory activities against α-glucosidase with IC50 values of 38.80 ± 1.01 μM, 32.32 ± 1.01 μM, and 75.20 ± 1.02 μM, respectively. Further pharmacological characterization revealed a mixed-mode inhibition for 5, a competitive inhibition for 7, while 11 acted as a non-competitive inhibitor.

Bioprospecting of Novel and Bioactive Metabolites from Endophytic Fungi Isolated from Rubber Tree Ficus elastica Leaves.

Endophytic fungi are an important component of plant microbiota, and have the excellent capacity for producing a broad variety of bioactive metabolites. These bioactive metabolites not only affect the survival of the host plant, but also provide valuable lead compounds for novel drug discovery. In this study, forty-two endophytic filamentous fungi were isolated from Ficus elastica leaves, and further identified as seven individual taxa by ITS-rDNA sequencing. The antimicrobial activity of these endophytic fungi was evaluated against five pathogenic microorganisms. Two strains, Fes1711 (Penicillium funiculosum) and Fes1712 (Trichoderma harzianum), displayed broad-spectrum bioactivities. Our following study emphasizes the isolation, identification and bioactivity testing of chemical metabolites produced by T. harzianum Fes1712. Two new isocoumarin derivatives (1 and 2), together with three known compounds (3-5) were isolated, and their structures were elucidated using NMR and MS. Compounds 1 and 2 exhibited inhibitory activity against Escherichia coli. Our findings reveal that endophytic fungi from the rubber tree F. elastica leaves exhibit unique characteristics and are potential producers of novel natural bioactive products.

High ophiobolin A production in endophytic fungus Bipolaris sp. associated with Datura metel

Ophiobolin A, a metabolite of fungi, is known to induce cell death and have anticancer activity. Therefore, obtaining ophiobolin A has become an important aspect in studying activity with medicinal properties that are affected by it. Ophiobolin A-producing filamentous fungi are endophytic or infectious microbes that attack annual and short-cycle plants. Here we isolated the endophyte of Datura metel, which is an annual plant that produces ophiobolin A. Results of this study have led to the identification of an endophytic filamentous fungus Bipolaris sp. with high ophiobolin A production (235 mg/L) in liquid culture after 21 days. Our findings further indicate that ophiobolin A-producing fungi live in short-cycle plants, and a method of finding the fungus is described.

Colonization of legumes by an endophytic Fusarium solani strain FsK reveals common features to symbionts or pathogens

Plant cellular responses to endophytic filamentous fungi are scarcely reported, with the majority of described colonization processes in plant-fungal interactions referring to either pathogens or true symbionts. Fusarium solani strain K (FsK) is a root endophyte of Solanum lycopersicum, which protects against root and foliar pathogens. Here, we investigate the association of FsK with two legumes (Lotus japonicus and Medicago truncatula) and report on colonization patterns and plant responses during the establishment of the interaction. L. japonicus plants colonized by FsK complete their life cycle and exhibit no apparent growth defects under normal conditions. We followed the growth of FsK within root-inoculated plants spatiotemporally and showed the capability of the endophyte to migrate to the stem. In a bipartite system comprising of the endophyte and either whole plants or root organ cultures, we studied the plant sub-cellular responses to FsK recognition, using optical, confocal and transmission electron microscopy. A polarized reorganization of the root cell occurs: endoplasmic reticulum/cytoplasm accumulation and nuclear placement at contact sites, occasional development of papillae underneath hyphopodia and membranous material rearrangements towards penetrating hyphae. Fungal hyphae proliferate within the vascular bundle of the plant. Plant cell death is involved in fungal colonization of the root. Our data suggest that the establishment of FsK within legume tissues requires fungal growth adaptations and plant cell-autonomous responses, known to occur during both symbiotic and pathogenic plant-fungal interactions. We highlight the overlooked plasticity of endophytic fungi upon plant colonization, and introduce a novel plant-endophyte association.

In vitro- exploration of Fungal Endophytes of Egyptian Cynara scolymus L. (artichoke) and Investigation of some their bioactive potentials

<p>Plant-associated microorganisms, especially endophytic fungi, represent an untapped resource for the discovery of biologically active natural products. The objectives of this study were to isolate, identify endophytic fungi, and produce their bioactive metabolites from the leaves of two varieties of Egyptian artichoke namely: French Hyrious and Egyptian Baladi. In addition, assess of their total antioxidant capacity (TAC), total phenolic content (TPC) and total flavonoid content (TFC). The results of this novel study show a total of 35 endophytic fungal species belonging to 14 genera were isolated from both artichoke leaves with gross total counts of colonizing endophytic fungi ranged from 71 to 123 cfu which is matching 78.89% to 136.67% of colonization frequency. All taxa recovered were assigned to Ascomycetes. In addition, there is high species richness and diversity indices of endophytic filamentous fungi in the leaves Baladi Artichoke as compared to its French rival.  <em>Alternaria alternata</em> were found to be the most frequently isolated dominant species. The TAC, TPC and TFC of the fungal cultures ranged from 163 to 681 mgAAE/gDW, 10.38 to 40.30 mgGAE/ gDW, and 13.92 to 173.55 mgQE/gDW, respectively. Furthermore, LC-ESI-MS/MS confirmed the presence of 1,3-dicaffeoylquinic acid and 1,5-dicaffeoylquinic acid in the methanolic extract of <em>A. alternata.</em> Hence, this novel study suggested that the metabolites produced by endophytic fungi associated with Egyptian artichoke could be explored as an economic and potential natural resources with diverse pharmaceutical and biological activities.</p>

Development of an efficient genetic system in a gene cluster-rich endophytic fungus Calcarisporium arbuscula NRRL 3705

Filamentous fungi are emerging as attractive producers of natural products with novel structures, diverse bioactivities and unprecedented enzymology. But their genetic systems are poorly developed, especially in some non-model endogenic fungi, which have hampered our genetic manipulation of their natural product development. Calcarisporium arbuscula NRRL 3705 is an endophytic filamentous fungus rich in biosynthetic gene clusters and primarily producing mycotoxin aurovertins. Here we optimized Agrobacterium tumefaciens-mediated transformation (ATMT)-based efficient DNA introduction into C. arbuscula. By complementation of the monooxygenase gene aurC in ΔaurC mutant as a model, we showed that a strong but down-regulated promoter aurAp and three strong constitutive promoter gpdAp, tef1p and tubCp could be used for gene overexpression. Meanwhile, red fluorescence protein (RFP) was expressed in this fungus under the control of tubCp, potentially paving the way for enzyme localization determination during natural product biosynthesis. Furthermore, we developed efficient and convenient gene disruption in C. arbuscula based on ATMT, as exemplified by deletion of aurA in ΔaurC mutant. Our efficiency of deletion ran at about 40%. These results suggest that ATMT-based transformation for gene ectopic expression or deletion is an efficient strategy for genetic manipulation of C. arbuscula, and can be readily adapted to other rare filamentous fungi, potentially to promote discovery and development of natural products.

Involvement of Dactylonectria and Ilyonectria spp. in tree decline affecting multi-generation apple orchards

This study started from typical replant disease symptoms limited to specific foci within three multi-generation orchards showing homogeneous growth. A plant growth assay was conducted using soil from symptomatic and asymptomatic tree root zones along planted rows and from strip-rows. Root colonizing fungal communities were investigated, then the study turned to pathogenicity of Cylindrocarpon-like fungi (Dactylonectria and Ilyonectria spp) and their extracellular exudates. Growth of apple rootstock plantlets in soil from symptomatic trees was significantly lower than in those observed in soil from asymptomatic trees and from strip-rows. Among the main group of endophytic filamentous fungi isolated from roots, Cylindrocapon-like fungi (Dactylonectria torresensis and, to a lesser extent, Ilyonectria robusta), along with binucleate Rhizoctonia spp., prevailed mostly in plantlets grown in soil collected from planted rows. On the other hand, Fusarium spp. prevailed in plantlets grown in soil from the strip-rows. Cylindrocarpon-like fungi was found to be the most negatively correlated with plant growth. As findings of artificial inoculation with main root colonizing fungal species were not in line with what was observed in native soils, a further investigation was performed on secondary metabolites through which Dactylonectria torresensis exerts pathogenicity; this analysis revealed that both phytotoxins (tentoxin, HC toxin and zearalenone) and cytotoxic compounds (rabelomycin and nidulin) may be involved. Findings suggest that extracellular compounds released by D. torresensis may have contributed to the severe growth reduction associated with replant disease-like symptoms.

Endophytic filamentous fungi from a Catharanthus roseus: Identification and its hydrolytic enzymes

This paper reported on the various filamentous fungi strains that were isolated from a wild grown Catharanthus roseus. Based on the morphological characteristics and molecular technique through a Polymerase Chain Reaction and DNA sequencing method using internal transcribed spacer (ITS), these fungi had been identified as a Colletotrichum sp., Macrophomina phaseolina, Nigrospora sphaerica and Fusarium solani. The ultrastructures of spores and hyphae were observed under a Scanning Electron Microscope. The hydrolytic enzyme test showed that all strains were positive in secreting cellulase. Colletotrichum sp. and F. solani strains also gave a positive result for amylase while only F. solani was capable to secrete protease. These fungi were putatively classified as endophytic fungi since they produced extracellular enzymes that allow them to penetrate plant cell walls and colonize with symbiotic properties.

Composition of the endophytic filamentous fungi associated with Cinchona ledgeriana seeds and production of Cinchona alkaloids.

Four kinds of endophytic filamentous fungi (code names: CLS-1, CLS-2, CLS-3, and CLS-4) associated with the seeds of Cinchona ledgeriana (Rubiaceae) from West Java, Indonesia, were isolated. All of the isolates were classified into Diaporthe spp. based on phylogenetic analysis of the nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) including the 5.8S ribosomal DNA region. All four of these endophytic fungi produce Cinchona alkaloids, mainly quinine and quinidine, in synthetic liquid medium.

Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae

The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48h at 22°C could lead to a relatively highest frequency of transformation, and 200μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast–Escherichia–Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae.

Diversity of endophytic fungi in Glycine max

Endophytic fungi are microorganisms that live within plant tissues without causing disease during part of their life cycle. With the isolation and identification of these fungi, new species are being discovered, and ecological relationships with their hosts have also been studied. In Glycine max, limited studies have investigated the isolation and distribution of endophytic fungi throughout leaves and roots. The distribution of these fungi in various plant organs differs in diversity and abundance, even when analyzed using molecular techniques that can evaluate fungal communities in different parts of the plants, such as denaturing gradient gel electrophoresis (DGGE). Our results show there is greater species richness of culturable endophytic filamentous fungi in the leaves G. max as compared to roots. Additionally, the leaves had high values for diversity indices, i.e. Simpsons, Shannon and Equitability. Conversely, dominance index was higher in roots as compared to leaves. The fungi Ampelomyces sp., Cladosporium cladosporioides, Colletotrichum gloeosporioides, Diaporthe helianthi, Guignardia mangiferae and Phoma sp. were more frequently isolated from the leaves, whereas the fungi Fusarium oxysporum, Fusarium solani and Fusarium sp. were prevalent in the roots. However, by evaluating the two communities by DGGE, we concluded that the species richness was higher in the roots than in the leaves. UPGMA analysis showed consistent clustering of isolates; however, the fungus Leptospora rubella, which belongs to the order Dothideales, was grouped among species of the order Pleosporales. The presence of endophytic Fusarium species in G. max roots is unsurprising, since Fusarium spp. isolates have been previously described as endophyte in other reports. However, it remains to be determined whether the G. max Fusarium endophytes are latent pathogens or non-pathogenic forms that benefit the plant. This study provides a broader knowledge of the distribution of the fungal community in G. max leaves and roots, and identifies the genetic relationships among the isolated species.

Enhancement of lipid productivity in oleaginous Colletotrichum fungus through genetic transformation using the yeast CtDGAT2b gene under model-optimized growth condition.

Oleaginous fungi are of special interest among microorganisms for the production of lipid feedstocks as they can be cultured on a variety of substrates, particularly waste lingocellulosic materials, and few fungal strains are reported to accumulate inherently higher neutral lipid than bacteria or microalgae. Previously, we have characterized an endophytic filamentous fungus Colletotrichum sp. DM06 that can produce total lipid ranging from 34% to 49% of its dry cell weight (DCW) upon growing with various carbon sources and nutrient-stress conditions. In the present study, we report on the genetic transformation of this fungal strain with the CtDGAT2b gene, which encodes for a catalytically efficient isozyme of type-2 diacylglycerol acyltransferase (DGAT) from oleaginous yeast Candida troplicalis SY005. Besides the increase in size of lipid bodies, total lipid titer by the transformed Colletotrichum (lipid content ∼73% DCW) was found to be ∼1.7-fold more than the wild type (lipid content ∼38% DCW) due to functional activity of the CtDGAT2b transgene when grown under standard condition of growth without imposition of any nutrient-stress. Analysis of lipid fractionation revealed that the neutral lipid titer in transformants increased up to 1.8-, 1.6- and 1.5-fold compared to the wild type when grown under standard, nitrogen stress and phosphorus stress conditions, respectively. Lipid titer of transformed cells was further increased to 1.7-fold following model-based optimization of culture conditions. Taken together, ∼2.9-fold higher lipid titer was achieved in Colletotrichum fungus due to overexpression of a rate-limiting crucial enzyme of lipid biosynthesis coupled with prediction-based bioprocess optimization.

Pachybasin, a Major Metabolite from Culture Broth of Endophytic Coelomyceteous AFKR-18 Fungus isolated from a Yellow Moonsheed Plant, Arcangelisia flava (L.) Merr.

Endophytic fungi have been known to produce a broad range of biologically active secondary metabolites. One endophytic filamentous fungus, Coelomycetes AFKR-18, isolated from the young stems of a yellow moonsheed plant, Arcangelisia flava, has been found to produce pachybasin when placed in a liquid medium. The chemical structure of pachybasin was deduced from MS, 1D-, 2D-NMR spectrum analysis, and from reference data. On a micro-dilution test, pachybasin showed antimicrobial activities against E. coli, B. subtilis, M. luteus, S. cerevisiae, C. albicans, A. niger, and A. flavus, with MIC values of 64.0 μg/mL, and against S. aureus and F. oxysporum with MIC values of 32.0 and 16.0 μg/mL respectively.

Ability of endophytic filamentous fungi associated with Cinchona ledgeriana to produce Cinchona alkaloids

We have investigated the ability of endophytic filamentous fungi associated with Cinchona ledgeriana (Rubiaceae) to produce Cinchona alkaloids on potato dextrose agar medium and in a synthetic liquid medium. It was found that all twenty-one endophytic fungi produce Cinchona alkaloids, despite their genetic differences.

A novel mitovirus from the hypogeous ectomycorrhizal fungus Tuber excavatum

The evolution of mycorrhizal mutualism had a profound impact on establishment of terrestrial life [19]. Fossil records indicate that the earliest land plants, which had no proper roots, were already colonized by hyphal fungi, which formed vesicles and arbuscules strikingly similar to modern vesicular-arbuscular mycorrhizas. Today, over 90% of plants form mycorrhizal associations [19]. Although saprotrophic fungi play a central role in the forest ecosystem by cycling, translocating and sequestrating decomposed material, mycorrhizal fungi render inorganic decomposed litter accessible to their plant hosts. In return for inorganics, they receive organic compounds, in particular from coniferous and deciduous trees [14, 19]. In spite of their ecological importance, ectomycorrhizal fungi have largely been ignored by virologists interested in studying mycoviruses [10, 20–22]. Such viruses infecting a broad range of fungi may show a high degree of specialization to their hosts, since some genera exclusively infect fungi, whereas others associated with certain virus families infect multior unicellular organisms [10]. Mycoviruses have been reported from almost the entire range of fungal phyla [9, 15, 17] and possess single-stranded (ssRNA) genomes (genera Botrexvirus, Mycoflexivirus; families Narnaviridae, Endornaviridae, Hypoviridae) [9, 11, 12] or doublestranded (dsRNA) genomes (families Barnaviridae, Birnaviridae, Chrysoviridae, Cystoviridae, Metaviridae, Partitiviridae, Pseudoviridae, Reoviridae and Totiviridae). Members of the Birnaviridae and Cystoviridae are reported to infect fungal hosts [9, 15, 21], but they are not listed to infect fungi in the current ICTV master species list 2009 (version 9; http://talk.ictvonline.org/files/ictv_documents/m/msl/1231.aspx) [7, 10]. Until recently, the family Narnaviridae contained viruses exclusively found in fungi [1]. Since Narnaviridae contains records recently added to the INSDC database of sequences derived from plant material (e.g., ‘grapevine associated narnavirus-1’; GenBank accession GU108586), an alternative explanation for these findings would be that these sequences were actually derived from endophytic filamentous fungi and yeast associated with the plant tissues, which has also been suggested and partially proven by Al Rwahnih et al. [1]. Moreover, given the fact that true plant-pathogenic viruses of the genus Ourmiavirus have been proposed to belong to the family Narnaviridae challanges the current classification of virus genera and the host specificity of members of the family Narnaviridae [18]. The genera Mitovirus and Narnavirus include the simplest mycoviruses, which lack coat proteins and are characterized by a single open reading frame (ORF) coding for a viral RNA-directed RNA polymerase (RdRp) [2–6, 16]. Mitoviruses are strictly associated with mitochondria and possess characteristic 50and 30-terminal untranslated regions (UTRs) of variable length, which are most likely responsible for replicase recognition [3, 4]. While most mitoviruses are found in plant-pathogenic fungi, such as Ophiostoma, Chrysoporthe or Botrytis, members of a small J. B. Stielow CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands