Do colonization by dark septate endophytes and elevated temperature affect pathogenicity of oomycetes?

Phialocephala subalpina is one of the most frequent dark septate root endophytes in tree roots but its function in forest ecosystems is largely unknown. A full-factorial infection experiment was performed, using six P.?subalpina isolates, two pathogenic oomycetes (Phytophthora plurivora [syn. Phytophthora citricola s.l.] and Elongisporangium undulatum [syn. Pythium undulatum]) and two temperature regimes (17.9 and 21.6?°C) to examine the ability of P.?subalpina to protect Norway spruce seedlings against root pathogens. Seedling survival, disease intensity and seedling growth were affected by P.?subalpina genotype, temperature and pathogen species. Some P.?subalpina isolates effectively reduced mortality and disease intensity caused by the two pathogens. Elevated temperature adversely affected seedling growth but did not aggravate the effect of the pathogens. Elongisporangium undulatum but not P.?plurivora significantly reduced plant growth. Colonization density of P.?subalpina measured by quantitative PCR was not affected by temperature or the presence of the pathogens. In conclusion, P.?subalpina confers an indirect benefit to its host and might therefore be tolerated in natural ecosystems, despite negative effects on plant health and plant growth.     

Resilience of Phialocephala fortinii s.l. – Acephala applanata communities – Effects of disturbance and strain introduction

Members of the Phialocephala fortinii s.l. – Acephala applanata species complex (PAC) are ubiquitous endophytes forming complex communities in roots of conifers and ericaceous shrubs across the Northern hemisphere. Two kinds of disturbances (clear-cutting and drought) and their effects on the resident PAC community, as well as on the introduction of alien strain 7_45_5, were investigated using mesocosms with natural Norway spruce (Picea abies) regeneration, both under controlled climate chamber conditions and natural conditions in the forest.This is the first record of successful strain introduction into a well-established forest ecosystem. Introduction was more successful when planting inoculated living spruce saplings, compared to inoculation using autoclaved colonized roots. 7_45_5 was less assertive in the forest, where Phialocephala subalpina clearly dominated. Clear-cutting favored A. applanata and simultaneously reduced the overall frequency of PAC. Drought only had a significant influence on 7_45_5, which was more abundant in dry than moist, non-clear-cut plots. To conclude, disturbances and arrival of foreign strains can alter resident PAC communities significantly.     

Isolation and characterization of microsatellite markers for the tree-root endophytes Phialocephala subalpina and Phialocephala fortinii s.s

Species of the Phialocephala fortinii s.l.-Acephala applanata complex are the dominant dark septate endophytes (DSE) in roots of species belonging to the Pinaceae. The two species Phialocephala subalpina and P. fortinii s.s. belong to the most widely distributed species within this complex. In the present study, 15 polymorphic microsatellite loci were developed for these two closely related species. Strains of a community which were analysed previously using single-copy restriction fragment length polymorphism were screened with the new markers. Microsatellites were suitable to classify the two species and to recognize individuals within species.     

Host species and strain combination determine growth reduction of spruce and birch seedlings colonized by root-associated dark septate endophytes

Interactions of Betula pendula and Picea abies with dark septate endophytes of the Phialocephala fortinii-Acephala applanata species complex (PAC) were studied. PAC are ubiquitous fungal root symbionts of many woody plant species but their ecological role is largely unknown. Sterile birch and spruce seedlings in monoculture and mixed culture were exposed to four PAC strains, added either singularly or paired in all possible combinations at 18°C and 23°C. Plant and fungal biomass was determined after 4 months. The most significant factors were strain and host combination. One of the strains significantly reduced biomass gain of spruce but not of birch. Plant biomass was negatively correlated with total endophytic fungal biomass in half of the strain - plant combinations. Endophytic PAC biomass was four times higher in spruce (≈ 40 mg g(-1) drw) than in birch (≈ 10 mg g(-1) drw). Competition between strains was strain-dependent with some strains significantly reducing colonization density of other strains, and, thus, attenuating adverse effects of 'pathogenic' strains on plant growth in some strain - plant combinations. Biomass gain of spruce but not of birch was significantly reduced at higher temperature. In conclusion, host, fungal genotype, colonization density and presence of a competing PAC strain were the main determining factors for plant growth.     

Inhibition of Phytophthora species by secondary metabolites produced by the dark septate endophyte Phialocephala europaea

Dark septate fungal root endophytes of the Phialocephala fortinii s.l.–Acephala applanata species complex (PAC) are widely distributed throughout the temperate and subtropical regions of the Northern Hemisphere. Previous studies have shown that some PAC members are pathogenic, others suppress oomycete root pathogens and some have no obvious effect on their Norway spruce (Picea abies) host. The activity of 85 PAC isolates against Phytophthora citricola s.l. was investigated by co-culture on plates. We identified a strain of Phialocephala europaea that significantly reduced the growth of P. citricola in vitro. Characterization of its extracellular metabolites resulted in the identification of four major compounds, sclerin, sclerolide, sclerotinin A, and sclerotinin B. These compounds are known for their positive as well as negative effects on plant growth. We found that sclerin and sclerotinin inhibited the growth of P. citricola in vitro at 150 μg ml^?1 (～1 mM). This is the first report of their production by Phialocephala and of activity of these compounds against an oomycete. Therefore, our data suggest that some PAC might reduce disease resulting from P. citricola by the production of antibiotics and plant growth promoting metabolites.     

Mycorrhiza Reduces Adverse Effects of Dark Septate Endophytes (DSE) on Growth of Conifers

Mycorrhizal roots are frequently colonized by fungi of the Phialocephala fortinii s.l. - Acephala applanata species complex (PAC). These ascomycetes are common and widespread colonizers of tree roots. Some PAC strains reduce growth increments of their hosts but are beneficial in protecting roots against pathogens. Nothing is known about the effects of PAC on mycorrhizal fungi and the PAC-mycorrhiza association on plant growth, even though these two fungal groups occur closely together in natural habitats. We expect reduced colonization rates and reduced negative effects of PAC on host plants if roots are co-colonized by an ectomycorrhizal fungus (ECM). Depending on the temperature regime interactions among the partners in this tripartite ECM-PAC-plant system might also change. To test our hypotheses, effects of four PAC genotypes (two pathogenic and two non-pathogenic on the Norway spruce), mycorrhization by Laccaria bicolor (strain S238N) and two temperature regimes (19°C and 25°C) on the biomass of the Douglas-fir (Pseudotsuga menziesii) and Norway spruce (Picea abies) seedlings were studied. Mycorrhization compensated the adverse effects of PAC on the growth of the Norway spruce at both temperatures. The growth of the Douglas-fir was not influenced either by PAC or mycorrhization at 19°C, but at 25°C mycorrhization had a similar protective effect as in the Norway spruce. The compensatory effects probably rely on the reduction of the PAC-colonization density by mycorrhizae. Temperature and the PAC strain only had a differential effect on the biomass of the Norway spruce but not on the Douglas-fir. Higher temperature reduced mycorrhization of both hosts. We conclude that ectomycorrhizae form physical and/or physiological barriers against PAC leading to reduced PAC-colonization of the roots. Additionally, our results indicate that global warming could cause a general decrease of mycorrhization making primary roots more accessible to other symbionts and pathogens.     

Assignment of species rank to six reproductively isolated cryptic species of the Phialocephala fortinii s.1.-Acephala applanata species complex

Phialocephala fortinii s.1. and Acephala applanata are the dominant dark septate endophytes (DSE) in roots of many trees and shrubs. Population genetic analysis led to the discovery of morphologically indistinguishable but reproductively isolated cryptic species (CSP) within Phialocephala fortinii s.1. In the present study we show that sequence data of two coding (beta-tubulin and translation elongation factor [EF-lalpha]) and three noncoding DNA loci confirm subdivision of P. fortinii s.1. and allow to differentiate seven CSP of P. fortinii. In addition we show that strains collected throughout Europe can be classified correctly based on these sequence markers. Statistically significant differences in growth response on different media were observed among CSP of P. fortinii and A. applanata. Growth inhibition on MEA amended with 100 mgl(-1) cycloheximide had the strongest differential effect of all physiological traits examined. In contrast exoenzyme production (laccase, proteinase, pectinase, phenol-oxidase, amylase, cytochrome oxidase and tyrosinase) rarely helped to differentiate CSP of P. fortinii. However A. applanata was a strong producer of amylases, laccases and proteinases. Based on these data we propose to assign species rank to six CSP of P. fortinii: P. turiciensis, P. letzii, P. europaea, P. helvetica, P. uotolensis, P. subalpina spp. nov. and P. fortinii s.s.     

Decomposition and Fungi of Needle Litter from Slow- and Fast-growing Norway Spruce (Picea abies) Clones

The fungal species involved in the decomposition of needle litter and their response to intraspecific genetic variation of trees are poorly known. First, we compared the needle decomposition and fungal decomposers underneath eight different Norway spruce clones in situ. This experiment revealed 60-70% loss of needle mass in two years. Although spruce clones differed considerably in growth (twofold height difference) and their needles differed in chemical composition, no significant difference was found for loss of needle mass under the spruce clones. Furthermore, the spruce clones did not affect the community structure of the fungal decomposers. Fungi inhabiting needle litter were identified by extracting ribosomal RNA (rRNA) and sequencing complementary DNA (cDNA) of internal trascribed spacer 1 (ITS1) region. The most frequent identifications were Lophodermium, Pezizales, Mycena, and Marasmius, suggesting that endophytic fungi were involved in the decomposition process. Second, we evaluated the potential of endophytes to decompose needle material in a microcosm experiment in which all other fungi than endophytes were excluded. Within 2 years, the endophytes had decomposed 35-45% of the needle mass. Sequences of Mollisia, Lophodermium, Lachnum, and Phialocephala were most frequently found in rRNA and rDNA extracted from the needles at the end of the microcosm experiment. The dominant needle endophyte in fresh, green needles was Lophodermium piceae, and this species was also found frequently in the needle material after 2 years of decay both in the field and laboratory experiments. Moreover, the relative abundance of Lophodermium-derived denaturing gradient gel electrophoresis (DGGE) bands correlated positively with the decomposition in the microcosm experiment. Hence, our results suggest a significant role of endophytic fungi, and particularly L. piceae, in the process of needle decomposition in boreal forests.     

Phylogeny of Phaeomollisia piceae gen. sp. nov.: a dark, septate, conifer-needle endophyte and its relationships to Phialocephala and Acephala

Dark, septate endophytes (DSE) were isolated from roots and needles of dwarf Picea abies and from roots of Vaccinium spp. growing on a permafrost site in the Jura Mountains in Switzerland. Two of the isolates sporulated after incubation for more than one year at 4 °C. One of them was a hitherto undescribed helotialean ascomycete Phaeomollisia piceae gen. sp. nov., the other was a new species of Phialocephala, P. glacialis sp. nov. Both species are closely related to DSE of the Phialocephala fortinii s. lat.-Acephala applanata species complex (PAC) as revealed by phylogenetic analyses of the ITS and 18S rDNA regions. Morphologically dissimilar fungi, such as Vibrissea and Loramyces species, are phylogenetically also closely linked to the new species and the PAC. Cadophora lagerbergii and C. (Phialophora) botulispora are moved to Phialocephala because Phialocephala dimorphospora and P. repens are the closest relatives. Several Mollisia species were closely related to the new species and the PAC according to ITS sequence comparisons. One DSE from needles of Abies alba and one from shoots of Castanea sativa formed Cystodendron anamorphs in culture. Their identical 18S sequences and almost identical ITS sequences indicated Mollisia species as closest relatives, suggesting that Mollisia species are highly euryoecious.     

The community of needle endophytes reflects the current physiological state of Norway spruce

This study investigated fungal endophytes in the needles of Norway spruce (Picea abies) cuttings in relation to host tree growth. We also determined the prevalence of endophytes in needles incubated for six months. The cuttings originated from clonal origins showing slow- and fast-growth in long-term field trials but the heritable differences in growth rate were not yet detected among the studied cutting. Endophytes were isolated from surface-sterilized needles with culture-free DNA techniques. No significant differences were observed between endophyte communities of slow- and fast-growing clonal origins. However, the endophyte community correlated with the current growth rate of cuttings suggesting that endophytes reflect short- rather than long-term performance of a host. The concentration of condensed tannins was similar in slow- and fast-growing clonal origins but it showed a negative relationship with endophyte species richness, implying that these secondary compounds may play an important role in spruce tolerance against fungal infections. More than a third of endophyte species were detected in both fresh and decomposing needles, indicating that many needle endophytes are facultative saprotrophs. Several potentially pathogenic fungal species were also found within the community of saprotrophic endophytes.     

Diversity of endophytic fungi of single Norway spruce needles and their role as pioneer decomposers

The diversity of endophytic fungi within single symptomless Norway spruce needles is described and their possible role as pioneer decomposers after needle detachment is investigated. The majority (90%) of all 182 isolates from green intact needles were identified as Lophodermium piceae. Up to 34 isolates were obtained from single needles. Generally, all isolates within single needles had distinct randomly amplified microsatellite (RAMS) patterns. Single trees may thus contain a higher number of L. piceae individuals than the number of their needles. To investigate the ability of needle endophytes to act as pioneer decomposers, surface-sterilized needles were incubated on sterile sand inoculated with autoclaved or live spruce forest humus layer. The dry weight loss of 13-17% found in needles after a 20-week incubation did not significantly differ between the sterilized and live treatments. Hence, fungi surviving the surface sterilization of needles can act as pioneer decomposers. A considerable portion of the needles remained green during the incubation. Brown and black needles, in which the weight loss had presumably taken place, were invaded throughout by single haplotypes different from L. piceae. Instead, Tiarasporella parca, a less common needle endophyte, occurred among these invaders of brown needles. Needle endophytes of Norway spruce seem thus to have different abilities to decompose host tissues after needle cast. L. piceae is obviously not an important pioneer decomposer of Norway spruce needles. The diversity of fungal individuals drops sharply when needles start to decompose. Thus, in single needles the decomposing mycota is considerably less diverse than the endophytic mycota.     

Population genetic analysis of Phialocephala fortinii s.l. and Acephala applanata in two undisturbed forests in Switzerland and evidence for new cryptic species

The genetic structure of the root endophytes Phialocephala fortinii s.l. and Acephala applanata was analyzed in two undisturbed forests. A total of 606 strains isolated from surface-sterilized, fine roots of Picea abies and Vaccinium myrtillus were examined. Two new cryptic species of P. fortinii were recognized and host specialization of A. applanata was confirmed. This species was almost exclusively isolated from roots of P. abies. The index of association did not deviate significantly from zero within any population, suggesting that recombination occurs or had occurred. Significant gene but no genotype flow was detected among study sites for P. fortinii s.l. In contrast, several isolates of A. applanata with both identical multi-locus haplotype and identical ISSR fingerprint were found in both study sites indicating genotype flow or a recent common history.     

The Potential of Dark Septate Endophytes to Form Root Symbioses with Ectomycorrhizal and Ericoid Mycorrhizal Middle European Forest Plants

The unresolved ecophysiological significance of Dark Septate Endophytes (DSE) may be in part due to existence of morphologically indistinguishable cryptic species in the most common Phialocephala fortinii s. l.—Acephala applanata species complex (PAC). We inoculated three middle European forest plants (European blueberry, Norway spruce and silver birch) with 16 strains of eight PAC cryptic species and other DSE and ectomycorrhizal/ericoid mycorrhizal fungi and focused on intraradical structures possibly representing interfaces for plant-fungus nutrient transfer and on host growth response. The PAC species Acephala applanata simultaneously formed structures resembling ericoid mycorrhiza (ErM) and DSE microsclerotia in blueberry. A. macrosclerotiorum, a close relative to PAC, formed ectomycorrhizae with spruce but not with birch, and structures resembling ErM in blueberry. Phialocephala glacialis, another close relative to PAC, formed structures resembling ErM in blueberry. In blueberry, six PAC strains significantly decreased dry shoot biomass compared to ErM control. In birch, one A. macrosclerotiorum strain increased root biomass and the other shoot biomass in comparison with non-inoculated control. The dual mycorrhizal ability of A. macrosclerotiorum suggested that it may form mycorrhizal links between Ericaceae and Pinaceae. However, we were unable to detect this species in Ericaceae roots growing in a forest with presence of A. macrosclerotiorum ectomycorrhizae. Nevertheless, the diversity of Ericaceae mycobionts was high (380 OTUs) with individual sites often dominated by hitherto unreported helotialean and chaetothyrialean/verrucarialean species; in contrast, typical ErM fungi were either absent or low in abundance. Some DSE apparently have a potential to form mycorrhizae with typical middle European forest plants. However, except A. applanata, the tested representatives of all hitherto described PAC cryptic species formed typical DSE colonization without specific structures necessary for mycorrhizal nutrient transport. A. macrosclerotiorum forms ectomycorrhiza with conifers but not with broadleaves and probably does not form common mycorrhizal networks between conifers with Ericaceae.     

Evidence for subdivision of the root-endophyte Phialocephala fortinii into cryptic species and recombination within species

The genetic structure of the root-endophyte Phialocephala fortinii was analyzed in three study sites using 11 single-copy RFLP probes. A total of 541 strains isolated from surface-sterilized, fine roots (diameter 0.5-3 mm) of Norway spruce (Picea abies) were examined. The average gene diversity (H) was high in all three study sites. Cluster analysis showed that up to four well-separated clusters of multi-locus haplotypes were present within the sites. Significant population subdivision was detected among these clusters, suggesting that groups of multi-locus haplotypes were reproductively isolated and that P. fortinii is a species complex composed of several cryptic species. This hypothesis was supported by ISSR-PCR which showed clusters consistent with those of the multi-locus haplotypes identified by RFLP analysis. In contrast, ITS sequence analysis did not allow to separate the species as clearly. The index of association (IA) did not deviate significantly from zero within any cryptic species, suggesting that recombination occurs within these species. Cryptic species occurred sympatrically. Thalli of two cryptic species were detected in the same 5-mm-long root segment in one instance. No significant differentiation was observed among populations of the same cryptic species in forest stands located approximately 5 km from each other. This finding is consistent with significant gene flow over this spatial scale. In addition, several isolates with both identical multi-locus haplotype and identical ISSR fingerprint were found at each study site indicating genotype flow or a recent common history between study sites.     

Microsatellite-Based Quantification Method to Estimate Biomass of Endophytic <Emphasis Type="Italic">Phialocephala</Emphasis> Species in Strain Mixtures

Fungi of the Phialocephala fortinii sensu lato–Acephala applanata species complex (PAC) are ubiquitous endophytic colonizers of tree roots in which they form genotypically diverse communities. Measurement of the colonization density of each of the fungal colonizers is a prerequisite to study the ecology of these communities. Up to now, there is no method readily available for the quantification of PAC strains co-colonizing the same root. The new DNA quantification method presented here is based on the amplification of microsatellites by competitive polymerase chain reaction (PCR). The method proved to be suitable to detect and quantify at least two strains within one single sample by the addition of a known amount of mycelium of a reference strain before DNA extraction. The method exploits the correlation between the reference/target ratio of light emitted during microsatellite detection (peak ratio) and the reference/target ratio of mycelial weights to determine the biomass of the target strain. Hence, calibration curves were obtained by linear regression of the peak ratios on the weight ratios for different mixtures of reference and target strains. The slopes of the calibration curves and the coefficients of determination were close to 1, indicating that peak ratios are good predictors of weight ratios. Estimates of fungal biomass in mycelial test mixtures of known composition laid within the 95% prediction interval and deviated on average by 16% (maximally 50%) from the true biomass. On average, 3–6% of the root biomass of Norway spruce seedlings consisted of mycelial biomass of either one of two inoculated PAC strains. Biomass estimates obtained by real-time quantitative PCR were correlated with the estimates obtained by the microsatellite-based method, but variation between the two estimates from the same root was high in some samples. The microsatellite-based DNA quantification method described here is currently the best method for strainwise estimation of endophytic biomass of PAC fungi in small root samples.     

Intraspecific variation in Heterobasidion annosum for mortality rate on Pinus sylvestris and Picea abies seedlings grown in pure culture

One-month-old Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) seedlings were inoculated in vitro with Heterobasidion annosum strains, four each of the P-, S- and F-intersterility groups. Variation among strains and between the IS groups in virulence, expressed in mortality rate, was detected during twelve months after inoculation. Most of the strains were more virulent on spruce than on pine, and mortality of spruce seedlings was significantly higher. The P strains displayed similar virulence on both hosts, while S strains caused higher mortality of spruce seedlings and significantly lower mortality of pine seedlings. Strains of the F group were less virulent, but killed significantly (P < 0.05) more spruce than pine seedlings. In the interspecific analyses with two hosts, the isolates and IS groups accounted for most of the explained variation in the host mortality.     

Characterisation of some Phlebiopsis gigantea isolates with respect to enzymatic activity and decay of Norway spruce wood

The activity of cellulase, peroxidase, phosphatase and dehydrogenase enzymes, together with the content of protocatechuic and vanillic acids, in samples of Norway spruce wood inoculated with 17 different isolates of Phlebiopsis gigantea was measured. The same isolates were used to compare decay activity in samples of Norway spruce wood after incubation for 3 and 6 months. Significant differences in enzyme activity and phenol production were found between aerial mycelium overgrowing the wood sample and the underlying wood. These differences indicated that the nature of the fungal mycelium appears to change depending on whether it is in contact with wood. After 6 months, highly extensive decomposition of the wood was shown by two British isolates. The results confirm a large difference in P. gigantea inoculum among isolates in natural conditions and reinforce the need for constant evaluation of the most active isolates to use in preparations for biocontrol: a problem for both users and registration bodies.     

Competitiveness of endophytic Phialocephala fortinii s.l. – Acephala applanata strains in Norway spruce roots

Dark septate endophytes of the Phialocephala fortinii s.l. – Acephala applanata species complex (PAC) are presumed to be the most abundant root colonizing endophytes of conifers across the Northern hemisphere. To test the competitiveness of different PAC strains, PAC-free Picea abies saplings were inoculated with five different PAC strains by planting them in pre-colonized substrates. Saplings were left to grow for six weeks and then transplanted crosswise into a substrate colonized by one of the other four strains for a further two weeks. PAC were isolated and genotyped using microsatellite markers. The power of colonization, i.e. the ability of colonizing roots already colonized by another PAC strain, and the power of retention, i.e. the ability of a resident strain of not being suppressed by an invading PAC strain, were calculated for each strain in every combination. The experiment was run twice under two different climatic conditions. Our results show that PAC strains differ (1) in their ability to colonize PAC-free, non-sterile roots, (2) in resistance against being suppressed by another PAC strain and (3) in their ability to invade roots already colonized by another PAC strain. In addition, both the PAC–PAC and the PAC-host interactions depend on the climatic conditions.     

Development of a nested PCR detection procedure for Nectria fuckeliana direct from Norway spruce bark extracts

A pair of primers specific for Nectria fuckeliana, a bark infecting pathogen predominantly of Norway spruce (Picea abies), were designed from comparisons of nucleotide sequences of the nuclear ribosomal internal transcribed spacer (ITS) regions of nine isolates from Norway, Lithuania, Switzerland, Austria, Slovakia, Scotland (Larix sp.) and New Zealand (Pinus radiata), and other closely related nectriaceous species, including Neo. Neomacrospora, and 'N'. mammoidea, to which it exhibits taxonomic similarities. Complete ITS sequence homology was observed between each of the nine N. fuckeliana isolates, regardless of geographic provenance, including a previously published Danish strain. Primers Cct1 and Cct2 consistently amplified a single product of 360 bp from DNA prepared from 20 isolates covering the principle range of the disease from Central and Northern Europe, but not from other Neonectria, 'Nectria' or a range of species commonly encountered in forest ecosystems, as well as P. abies or P. radiata DNA. A quick, simple and efficient mechanical lysis procedure for the extraction of high quality total DNA from bark, coupled with post-extraction polyvinylpolypyrrolidone (PVPP) chromatography purification, is described to facilitate successful PCR detection of N. fuckeliana direct from bark extracts. Detection of N. fuckeliana from bark preparations was only possible following nested PCR of PVPP purified extracts using universal primers ITS5 and 4 in first round amplification. The identity of products from bark tissues was confirmed by Southern hybridisation and sequencing. Using the above procedure, positive diagnosis of N. fuckeliana was achievable within 5 h and has the potential for full exploitation as both a forest management and ecological research tool. As the DNA extraction procedure described here has been successful in application against other tree species, it has potential for incorporation into other molecular diagnostic systems for other microorganisms responsible for other wood or tree bark diseases.     

Colonization of roots of cultivated Solanum lycopersicum by dark septate and other ascomycetous endophytes

Tomato (Solanum lycopersicum L.) roots from four different crop sites in Colombia were surface sterilized and 51 fungal isolates were obtained and conserved for further analysis. Based on microscopical observations and growth characteristics, 20 fungal isolates corresponded to genus Fusarium, six presented asexual conidia different from Fusarium, eight were sterile mycelia, seven of which had dark septate hyphae and 17 did not continue to grow on plates after being recovered from conservation. Growth on different media, detailed morphological characterization and ITS region sequencing of the six sporulating and eight sterile isolates revealed that they belonged to different orders of Ascomycota and that the sterile dark septate endophytes did not correspond to the well known Phialocephala group. Interactions of nine isolates with tomato plantlets were assessed in vitro. No effect on shoot development was revealed, but three isolates caused brown spots in roots. Colonization patterns as analyzed by confocal microscopy differed among the isolates and ranged from epidermal to cortical penetration. Altogether 11 new isolates from root endophytic fungi were obtained, seven of which showed features of dark septate endophytes. Four known morphotypes were represented by five isolates, while six isolates belonged to five morphotypes of putative new unknown species.