Christoph R. Grünig

Reproductive mode and life cycle of the ash dieback pathogen Hymenoscyphus pseudoalbidus.

Ash dieback caused by the fungal pathogen Hymenoscyphus pseudoalbidus is currently ravaging in Europe, killing Fraxinus excelsior and Fraxinus angustifolia trees of all age classes. The aim of this work was to elucidate aspects of the reproduction biology of this fungal pathogen and its cryptic, non-pathogenic sister species Hymenoscyphus albidus. The mating type (MAT) locus of both species was identified, partly sequenced and characterized. Whereas a heterothallic MAT organization was detected in H. pseudoalbidus, H. albidus was shown to be structurally homothallic. The molecular MAT determination of H. pseudoalbidus was confirmed by crossing experiments on sterile ash petioles. Crossings of strains exhibiting alternate MAT idiomorphs produced fertile apothecia whereas crosses of strains with identical MAT idiomorphs were never successful. Offspring genotyping with microsatellites (MSs) and the MAT marker confirmed that both parental strains were involved in apothecia formation. In addition, polymorphic MS were shown to follow Mendelian inheritance. However, for yet unknown reasons the MAT ratio of progenies of one successful cross revealed a significant segregation distortion. Based on the MAT sequences of H. pseudoalbidus a multiplex PCR was developed, allowing for a quick and reliable MAT determination. The PCR was applied to screen the MAT ratio of two H. pseudoalbidus populations derived from the country of the disease outbreak in Poland and two populations from the disease periphery in Switzerland. None of the screened populations showed a significant deviation from the 1:1 ratio, expected under random mating. Therefore, an initial clonal distribution through asexually produced conidiospores as observed for other fungal pathogens holds not true for H. pseudoalbidus. Instead, our data is highly supportive for a distribution through ascospores. Leaf petioles collected in the field were thoroughly analyzed for the number of different colonizing strains and their mating behavior. Up to eight different H. pseudoalbidus genotypes were found on a single petiole. Cross-fertilizations of strains on the same petiole and fertilizations of unknown strains from outside were found, indicating that fertilization is mediated by spermatia. The presented study complements our understanding of the life cycle of this highly destructive pathogen. The possibility to perform sexual crosses in the lab provides ample opportunities for further genetic studies of H. pseudoalbidus and related species in the future.

Molecular and phenotypic description of the widespread root symbiont Acephala applanata gen. et sp. nov., formerly known as dark-septate endophyte Type 1

Acephala applanata gen. et sp. nov. is described. A. applanata is a dark-septate endophyte (DSE) of conifer roots and belongs to the Phialocephala fortinii species complex. Several genetic markers, including isozymes, inter-simple-sequence-repeat (ISSR) fingerprints, single-copy restriction fragment length polymorphisms (RFLP) and sequences of the internal transcribed spacers (ITS), let us unambiguously separate isolates of A. applanata from isolates of P. fortinii s.l. and other dark-septate endophytes. Alleles at four RFLP loci and two fixed nucleotides in the ITS region were diagnostic for A. applanata. One of the fixed nucleotides resulted in the addition of an Afa I restriction site. PCR amplification with primers prITS4 and the newly developed primer PF-ITS_F (ACT CTG AAT GTT AGT GAT GTC TGA GT) and restriction digestion with Afa I yielded three fragments (203 bp, 117 bp, 56 bp) in A. applanata but only two (260 bp and 117 bp) in P. fortinii s.l. Population differentiation (GST) between A. applanata and other cryptic species of P fortinii was pronounced, and the index of association (IA) did not deviate significantly from zero, showing that recombination occurs or had occurred in A. applanata. Although isolates of A. applanata never were observed to sporulate, it can be distinguished morphologically from P fortinii s.l. by the scarcity of aerial mycelium, significantly slower growth and denser mycelium on cellophane overlaid on water agar. These phenotypic characteristics, combined with diagnostic RFLP alleles and/or PCR-RFLP of the ITS fragment with the fixed Afa I restriction site, unequivocally allow identification of A. applanata.

Negative effects on survival and performance of Norway spruce seedlings colonized by dark septate root endophytes are primarily isolate-dependent

Root endophytes are common and genetically highly diverse suggesting important ecological roles. Yet, relative to above-ground endophytes, little is known about them. Dark septate endophytic fungi of the Phialocephala fortinii s.l.-Acephala applanata species complex (PAC) are ubiquitous root colonizers of conifers and Ericaceae, but their ecological function is largely unknown. Responses of Norway spruce seedlings of two seed provenances to inoculations with isolates of four PAC species were studied in vitro. In addition, isolates of Phialocephala subalpina from two populations within and one outside the natural range of Norway spruce were also included to study the effect of the geographic origin of P. subalpina on host response. The interaction of PAC with Norway spruce ranged from neutral to highly virulent and was primarily isolate-dependent. Variation in virulence was much higher within than among species, nonetheless only isolates of P. subalpina were highly virulent. Disease caused by P. subalpina genotypes from the native range of Norway spruce was more severe than that induced by genotypes from outside the natural distribution of Norway spruce. Virulence was not correlated with the phylogenetic relatedness of the isolates but was positively correlated with the extent of fungal colonization as measured by quantitative real-time PCR.

Spatial distribution of dark septate endophytes in a confined forest plot

In the present study we investigated the abundance and spatial distribution of dark septate root endophytes (DSE) in a 3 x 3 m plot in a spruce stand ( Picea abies ). A total of 144 DSE isolates were obtained by means of a hierarchical sampling design. Most roots were colonised, as DSE were isolated from 81.7% of root segments. ISSR-PCR fingerprinting was used to identify 21 unique ISSR types. Dominant types were isolated from adjacent points that covered an area of up to 6.8 m 2 of the study plot, and ISSR types were intermingled extensively. Frequency of isolation of the different ISSR types was uneven with two dominant types that accounted for 38% and 28% of all DSE isolates, respectively. Seven DSE strains representing six different ISSR types were identified as Phialocephala fortinii based on the morphology of fertile conidiophores and/or ITS 1 and 2 sequence comparisons.

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 degrees 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.

Characterization of Guignardia mangiferae isolated from tropical plants based on morphology, ISSR-PCR amplifications and ITS1-5.8S-ITS2 sequences

Phenotypic and genotypic variability of 18 endophytic Guignardia strains from different host plants (Anacardiaceae: Anacardium giganteum, Myracrodruon urundeuva, Spondias mombin; Apocynaceae: Aspidosperma polyneuron; Ericaceae: Rhododendron sp.; Fabaceae: Bowdichia nitida; Leguminosae: Cassia occidentalis; Rutaceae: Citrus aurantium) growing in different sites in Brazil was assessed by means of morphometric measurements and inter-single-sequence-repeat-anchored polymerase chain reaction (ISSR-PCR) amplifications of the DNA. Morphology of conidia and ascospores and growth rates of the Brazilian isolates corresponded well with those of G. mangiferae. Multiple correspondence analysis (MCA) of the ISSR-PCR data yielded three groups of strains, which did, however, not correspond either to the host or to the geographic origin. The same individual tree hosted genotypically different strains indicating multiple infections. Phylogeny based on ITS1-5.8S-ITS2 sequence analyses confirmed conspecificity of the Brazilian isolates with G. mangiferae (syn. G. endophyllicola).

Development of single-copy RFLP markers for population genetic studies of Phialocephala fortinii and closely related taxa

Single-copy RFLP markers were developed for the root endophytic fungus Phialocephala fortinii. After an initial screening of 40 probes with four restriction enzymes, EcoRV and HindIII were chosen to analyse 31 strains of P. fortinii and nine strains of a closely related morphotype (Type 1) which have known ISSR-PCR or allozyme phenotypes. Type 1 isolates and a single genetically different isolate with Type 1 like morphology, a representative of a possible third taxon, showed unique alleles with several probes and both restriction enzymes. Consequently, both taxa were easily distinguishable from P. fortinii isolates. Whereas EcoRV in combination with seven probes revealed only eight multi-locus haplotypes among 17 ISSR phenotypes, HindIII in combination with 12 probes was able to distinguish all ISSR and/or allozyme phenotypes except two pairs of strains. Strains of P. fortinii showed a high gene diversity, and up to 15 alleles per locus were observed among the 31 strains of P. fortinii. The 12 probes used in combination with HindIII were (partially) sequenced and BLAST searches showed no similarities with known sequences indicating that they probably are non-coding regions of the genome. Enzyme-probe combinations suitable to indicate the genetic structure of P. fortinii and Type 1 populations were easily identified in the present study. This opens up avenues to study communities of the P. fortinii complex exposed to various environments and management intensities, and will help to promote the understanding of these extremely successful organisms.

Suitability of quantitative real-time PCR to estimate the biomass of fungal root endophytes.

ABSTRACT A nested single-copy locus-based quantitative PCR (qPCR) assay and a multicopy locus-based qPCR assay were developed to estimate endophytic biomass of fungal root symbionts belonging to the Phialocephala fortinii sensu lato-Acephala applanata species complex (PAC). Both assays were suitable for estimation of endophytic biomass, but the nested assay was more sensitive and specific for PAC. For mycelia grown in liquid cultures, the correlation between dry weight and DNA amount was strong and statistically significant for all three examined strains, allowing accurate prediction of fungal biomass by qPCR. For mycelia colonizing cellophane or Norway spruce roots, correlation between biomass estimated by qPCR and microscopy was strain dependent and was affected by the abundance of microsclerotia. Fungal biomass estimated by qPCR and microscopy correlated well for one strain with poor microsclerotia formation but not for two strains with high microsclerotia formation. The accuracy of qPCR measurement is constrained by the variability of cell volumes, while the accuracy of microscopy can be hampered by overlapping fungal structures and lack of specificity for PAC. Nevertheless, qPCR is preferable because it is highly specific for PAC and less time-consuming than quantification by microscopy. There is currently no better method than qPCR-based quantification using calibration curves obtained from pure mycelia to predict PAC biomass in substrates. In this study, the DNA amount of A. applanata extracted from 15 mm of Norway spruce fine root segments (mean diameter, 610 μm) varied between 0.3 and 45.5 ng, which corresponds to a PAC biomass of 5.1 ± 4.5 μg (estimate ± 95% prediction interval) and 418 ± 264 μg.

Microsatellite size homoplasies and null alleles do not affect species diagnosis and population genetic analysis in a fungal species complex

The suitability of 13 microsatellite loci for species diagnosis and population genetics in 11 species of the Phialocephala fortinii s.l.–Acephala applanata species complex (PAC) was assessed. Two data sets were compared to test possible biases in species typing and clone detection resulting from null alleles and size homoplasies. The first data set was based on fragment lengths derived from a multiplex polymerase chain reaction (PCR) assay and the second data set was received from singleplex PCR at lower stringency and sequencing. Most null alleles observed in the multiplex PCR assay could be amplified during singleplex PCR under less stringent conditions. Size homoplasies resulting from mutations in flanking regions and differences in microsatellite structures were observed. For example, Phialocephala uotolensis possessed a (CT)13 in addition to the (GT)x motif at locus mPF_0644. Despite the occurrence of null alleles and size homoplasies, species diagnosis and population genetic analysis studies were not affected. These markers will facilitate studies on population biology, ecology and biogeography of PAC species.

Characterization of the mating type (MAT) locus in the Phialocephala fortinii s.l. - Acephala applanata species complex.

Members of the Phialocephala fortinii sensu lato -Acephala applanata species complex (PAC) are the dominant root endophytes of woody plants in temperate and boreal forests. In the present study, the mating type (MAT) idiomorphs of eight species belonging to the PAC were cloned. Because direct cloning of MAT idiomorphs was not possible, species phylogenetically placed between the PAC and other helotialean species with characterized MAT locus were used for an intermediate cloning step. Whereas A. applanata showed a homothallic organization structure of the MAT locus, all other species either contained the MAT1-1 or MAT1-2 idiomorph indicative of heterothallism. A Tc1-like transposable element was found within the MAT locus of A. applanata. Analysis of A. applanata strains collected over a broad geographical range showed that the transposable element was present in all A. applanata strains, suggesting an ancient transposition event. Moreover, a partial MAT1-1-1 gene was identified within MAT1-2 idiomorphs, a common phenomenon in the order Helotiales. However, this partial gene was not fixed in all populations of the species. The evolution of the MAT locus with regard to different mating systems is discussed for the species complex.