Development and implementation of a novel CAPS assay reveals high prevalence of a boscalid resistance marker and its co-occurrence with an azole resistance marker in Erysiphe necator.

Succinate dehydrogenase inhibitors (SDHIs), are frequently used against powdery mildew (PM) fungi, such as Erysiphe necator, the causal agent of grapevine PM. Fungicide resistance, however, hinders effective control. DNA-based monitoring facilitates the recognition of resistance. We aimed (i) to adapt an effective method to detect a widespread genetic marker of resistance to boscalid, a commonly used SDHI, and (ii) to study the co-occurrence of the marker with a marker of resistance to demethylase inhibitor (DMI) fungicides. Sequencing of the sdhB gene identified a non-synonymous substitution, denoted as sdhB-A794G, leading to an amino acid change (H242R) in the sdhB protein. In vitro fungicide resistance tests showed that E. necator isolates carrying sdhB-A794G were resistant to boscalid. We adopted a cleaved amplified polymorphic sequence-based method and screened more than 500 field samples collected from five Hungarian wine regions in two consecutive years. The sdhB-A794G marker was detected in all wine regions and in both years, altogether in 61.7% of samples, including 20.5% in which both sdhB-A794G and the wild-type were present. The frequency of sdhB-A794G was higher in SDHI-treated vineyards than in vineyards without any SDHI application. A significant difference in the presence of the marker was detected among wine regions; its prevalence ranged from none to 100%. We identified significant co-occurrence of sdhB-A794G with the CYP51-A495T (Y136F) mutation of the CYP51 gene, a known marker of resistance to DMIs. The monitoring of fungicide resistance is fundamental for the successful control of E. necator. Our rapid, cost-effective diagnostic method will support decision-making and fungicide resistance monitoring and management.

Trans-kingdom fungal pathogens infecting both plants and humans, and the problem of azole fungicide resistance.

Azole antifungals are abundantly used in the environment and play an important role in managing fungal diseases in clinics. Due to the widespread use, azole resistance is an emerging global problem for all applications in several fungal species, including trans-kingdom pathogens, capable of infecting plants and humans. Azoles used in agriculture and clinics share the mode of action and facilitating cross-resistance development. The extensive use of azoles in the environment, e.g., for plant protection and wood preservation, contributes to the spread of resistant populations and challenges using these antifungals in medical treatments. The target of azoles is the cytochrome p450 lanosterol 14-α demethylase encoded by the CYP51 (called also as ERG11 in the case of yeasts) gene. Resistance mechanisms involve mainly the mutations in the coding region in the CYP51 gene, resulting in the inadequate binding of azoles to the encoded Cyp51 protein, or mutations in the promoter region causing overexpression of the protein. The World Health Organization (WHO) has issued the first fungal priority pathogens list (FPPL) to raise awareness of the risk of fungal infections and the increasingly rapid spread of antifungal resistance. Here, we review the main issues about the azole antifungal resistance of trans-kingdom pathogenic fungi with the ability to cause serious human infections and included in the WHO FPPL. Methods for the identification of these species and detection of resistance are summarized, highlighting the importance of these issues to apply the proper treatment.

Exploring diversity within the genus Tulostoma (Basidiomycota, Agaricales) in the Pannonian sandy steppe: four fascinating novel species from Hungary.

Steppe vegetation on sandy soil in Hungary has recently been revealed as one of the hot spots in Europe for the stalked puffballs (genus Tulostoma). In the framework of the taxonomic revision of gasteroid fungi in Hungary, four Tulostoma species are described here as new to science: T.dunense, T.hungaricum, T.sacchariolens and T.shaihuludii. The study is based on detailed macro- and micromorphological investigations (including light and scanning electron microscopy), as well as a three-locus phylogeny of nrDNA ITS, nrDNA LSU and tef1-α sequences. The ITS and LSU sequences generated from the type specimen of T.cretaceum are provided and this resolved partly the taxonomy of the difficult species complex of T.aff.cretaceum.

Untargeted metabolomic analyses support the main phylogenetic groups of the common plant-associated Alternaria fungi isolated from grapevine (Vitis vinifera).

Alternaria, a cosmopolitan fungal genus is a dominant member of the grapevine (Vitis vinifera) microbiome. Several Alternaria species are known to produce a variety of secondary metabolites, which are particularly relevant to plant protection and food safety in field crops. According to previous findings, the majority of Alternaria species inhabiting grapevine belong to Alternaria sect. Alternaria. However, the phylogenetic diversity and secondary metabolite production of the distinct Alternaria species has remained unclear. In this study, our aim was to examine the genetic and metabolic diversity of endophytic Alternaria isolates associated with the above-ground tissues of the grapevine. Altogether, 270 Alternaria isolates were collected from asymptomatic leaves and grape clusters of different grapevine varieties in the Eger wine region of Hungary. After analyses of the nuclear ribosomal DNA internal transcribed spacer (ITS) and RNA polymerase second largest subunit (rpb2) sequences, 170 isolates were chosen for further analyses. Sequences of the Alternaria major allergen gene (Alt a 1), endopolygalacturonase (endoPG), OPA10-2, and KOG1058 were also included in the phylogenetic analyses. Identification of secondary metabolites and metabolite profiling of the isolates were performed using high-performance liquid chromatography (HPLC)-high-resolution tandem mass spectrometry (HR-MS/MS). The multilocus phylogeny results revealed two distinct groups in grapevine, namely A. alternata and the A. arborescens species complex (AASC). Eight main metabolites were identified in all collected Alternaria isolates, regardless of their affiliation to the species and lineages. Multivariate analyses of untargeted metabolites found no clear separations; however, a partial least squares-discriminant analysis model was able to successfully discriminate between the metabolic datasets from isolates belonging to the AASC and A. alternata. By conducting univariate analysis based on the discriminant ability of the metabolites, we also identified several features exhibiting large and significant variation between A. alternata and the AASC. The separation of these groups may suggest functional differences, which may also play a role in the functioning of the plant microbiome.

Comprehensive analyses of the occurrence of a fungicide resistance marker and the genetic structure in Erysiphe necator populations.

Genetically distinct groups of Erysiphe necator, the fungus causing grapevine powdery mildew infect grapevine in Europe, yet the processes sustaining stable genetic differences between those groups are less understood. Genotyping of over 2000 field samples from six wine regions in Hungary collected between 2017 and 2019 was conducted to reveal E. necator genotypes and their possible differentiation. The demethylase inhibitor (DMI) fungicide resistance marker A495T was detected in all wine regions, in 16% of the samples. Its occurrence differed significantly among wine regions and grape cultivars, and sampling years, but it did not differ between DMI-treated and untreated fields. Multilocus sequence analyses of field samples and 59 in vitro maintained isolates revealed significant genetic differences among populations from distinct wine regions. We identified 14 E. necator genotypes, of which eight were previously unknown. In contrast to the previous concept of A and B groups, European E. necator populations should be considered genetically more complex. Isolation by geographic distance, growing season, and host variety influence the genetic structuring of E. necator, which should be considered both during diagnoses and when effective treatments are planned.

Impact of dark septate endophytes on salt stress alleviation of tomato plants.

Fungal endophytes can improve plant tolerance to abiotic stress conditions. Dark septate endophytes (DSEs) belong to phylogenetically non-related groups of root colonizing fungi among the Ascomycota with high melanin-producing activities. They can be isolated from roots of more than 600 plant species in diverse ecosystems. Still the knowledge about their interaction with host plants and their contribution to stress alleviation is limited. The current work aimed to test the abilities of three DSEs (Periconia macrospinosa, Cadophora sp., Leptodontidium sp.) to alleviate moderate and high salt stress in tomato plants. By including an albino mutant, the role of melanin for the interaction with plants and salt stress alleviation could also be tested. P. macrospinosa and Cadophora sp. improved shoot and root growth 6 weeks after inoculation under moderate and high salt stress conditions. No matter how much salt stress was applied, macroelement (P, N, and C) contents were unaffected by DSE inoculation. The four tested DSE strains successfully colonized the roots of tomato, but the colonization level was clearly reduced in the albino mutant of Leptodontidium sp. Any difference in the effects on plant growth between the Leptodontidium sp. wild type strain and the albino mutant could, however, not be observed. These results show that particular DSEs are able to increase salt tolerance as they promote plant growth specifically under stress condition. Increased plant biomasses combined with stable nutrient contents resulted in higher P uptake in shoots of inoculated plants at moderate and high salt conditions and higher N uptake in the absence of salt stress in all inoculated plants, in P. macrospinosa-inoculated plants at moderate salt condition and in all inoculated plants except the albino mutants at high salt condition. In summary, melanin in DSEs seems to be important for the colonization process, but does not influence growth, nutrient uptake or salt tolerance of plants.

Arylnaphthalene Lignans with Anti-SARS-CoV-2 and Antiproliferative Activities from the Underground Organs of Linum austriacum and Linum perenne.

Diphyllin (1) and justicidin B (2) are arylnaphthalene lignans with antiviral and antiproliferative effects. Compound 1 is also known as an effective inhibitor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). To evaluate the in vitro antiviral and cytotoxic potency of both lignans in SARS-CoV-2 -infected cells and various cancer cell lines, respectively, 1 and 2 were isolated from the underground organs of Linum austriacum and Linum perenne. Two previously undescribed arylnaphthalene lignans, denominated linadiacin A and B (3 and 4), were also isolated and identified. In acidic media, 3 was converted by a two-step reaction into 2 via the intermediate 4. Optimum acid treatment conditions were determined to isolate lignans by one-step preparative high-performance liquid chromatography (HPLC). The results of the conversion, HPLC-tandem mass spectrometry, nuclear magnetic resonance spectroscopy, and molecular modeling studies allowed complete structure analysis. Compounds 1 and 2 were the most effective against SARS-CoV-2 with a 3-log reduction in the viral copy number at a 12.5 µM concentration. Ten human cancer cell lines showed sensitivity to at least one of the isolated lignans.

Label-free biosensing of lignans for therapeutics using engineered model surfaces.

The label-free interaction analysis of macromolecules and small molecules has increasing importance nowadays, both in diagnostics and therapeutics. In the blood vascular system, human serum albumin (HSA) is a vital globular transport protein with potential multiple ligand binding sites. Characterizing the binding affinity of compounds to HSA is essential in pharmaceutics and in developing new compounds for clinical application. Aryltetralin lignans from the roots of Anthriscus sylvestris are potential antitumor therapeutic candidates, but their molecular scale interactions with specific biomolecules are unrevealed. Here, we applied the label-free grating-coupled interferometry (GCI) biosensing method with a polycarboxylate-based hydrogel layer with immobilized HSA on top of it. With this engineered model surface, we could determine the binding parameters of two novel aryltetralin lignans, deoxypodophyllotoxin (DPT), and angeloyl podophyllotoxin (APT) to HSA. Exploiting the multi-channel referencing ability, the unique surface sensitivity, and the throughput of GCI, we first revealed the specific biomolecular interactions. Traditional label-free kinetic measurements were also compared with a novel, fast way of measuring affinity kinetics using less sample material (repeated analyte pulses of increasing duration (RAPID)). Experiments with well-characterized molecular interactions (furosemide to carbonic-anhydrase (CAII) and warfarin, norfloxacin to HSA) were performed to prove the reliability of the RAPID method. In all investigated cases, the RAPID and traditional measurement gave similar affinity values. In the case of DPT, the measurements and relevant modeling suggested two binding sites on HSA, with dissociation constant values of Kd1 = 1.8 ± 0.01 µM, Kd2 = 3 ± 0.02 µM. In the case of APT, the experiments resulted in Kd1 = 9 ± 1.7 µM, Kd2 = 28 ± 0.3 µM. The obtained binding values might suggest the potential medical application of DPT and APT without further optimization of their binding affinity to HSA. These results could be also adapted to other biomolecules and applications where sample consumption and the rapidity of the measurements are critical.

Multilocus molecular phylogenetic-led discovery and formal recognition of four novel root-colonizing Fusarium species from northern Kazakhstan and the phylogenetically divergent Fusarium steppicola lineage.

In this study, DNA sequence data were used to characterize 290 Fusarium strains isolated during a survey of root-colonizing endophytic fungi of agricultural and nonagricultural plants in northern Kazakhstan. The Fusarium collection was screened for species identity using partial translation elongation factor 1-α (TEF1) gene sequences. Altogether, 16 different Fusarium species were identified, including eight known and four novel species, as well as the discovery of the phylogenetically divergent F. steppicola lineage. Isolates of the four putatively novel fusaria were further analyzed phylogenetically with a multilocus data set comprising partial sequences of TEF1, RNA polymerase II largest (RPB1) and second-largest (RPB2) subunits, and calmodulin (CaM) to assess their genealogical exclusivity. Based on the molecular phylogenetic and comprehensive morphological analyses, four new species are formally described herein: F. campestre, F. kazakhstanicum, F. rhizicola, and F. steppicola.

Darksidea phi, sp. nov., a dark septate root-associated fungus in foundation grasses in North American Great Plains.

Darksidea is a common genus of dark septate fungi-a group of ascomycetes in semiarid regions. A survey reported D. alpha and a distinct Darksidea lineage as abundant root-associated fungi of foundational grasses in North America. Fungi were isolated, and metabarcode data were obtained from sequencing of fungal communities of grass roots in the United States. During a comprehensive investigation of the Darksidea lineage, we carried out polyphasic taxonomy, genomic characterization, and identification of host associations, geographic distribution, and environmental factors that correlate with its abundance. For molecular phylogenetic studies, seven loci were sequenced. Isolates of the distinct Darksidea had variable colony morphology. No sexual reproductive structures were detected, but chlamydospores were frequently observed. The complete genome of an isolate of the lineage was sequenced with a size of 52.3 Mb including 14 707 gene models. Based on morphology and phylogenetic analysis, we propose the novel species Darksidea phi, sp. nov. Metabarcoding data showed that D. phi distribution and relative abundance were not limited to semiarid regions or a specific grass species, suggesting low host specificity among graminoids. This new species, D. phi, expands the distribution of the genus in the United States beyond prior reports from arid regions.

Micro-scale Experimental System Coupled with Fluorescence-based Estimation of Fungal Biomass to Study Utilisation of Plant Substrates.

The degradation capacity and utilisation of complex plant substrates are crucial for the functioning of saprobic fungi and different plant symbionts with fundamental functions in ecosystems. Measuring the growth capacity and biomass of fungi on such systems is a challenging task. We established a new micro-scale experimental setup using substrates made of different plant species and organs as media for fungal growth. We adopted and tested a reliable and simple titration-based method for the estimation of total fungal biomass within the substrates using fluorescence-labelled lectin. We found that the relationship between fluorescence intensity and fungal dry weight was strong and linear but differed among fungi. The effect of the plant organ (i.e. root vs. shoot) used as substrate on fungal growth differed among plant species and between root endophytic fungal species. The novel microscale experimental system is useful for screening the utilisation of different substrates, which can provide insight into the ecological roles and functions of fungi. Furthermore, our fungal biomass estimation method has applications in various fields. As the estimation is based on the fungal cell wall, it measures the total cumulative biomass produced in a certain environment.

Natural Compounds as Target Biomolecules in Cellular Adhesion and Migration: From Biomolecular Stimulation to Label-Free Discovery and Bioactivity-Based Isolation.

Plants and fungi can be used for medical applications because of their accumulation of special bioactive metabolites. These substances might be beneficial to human health, exerting also anti-inflammatory and anticancer (antiproliferative) effects. We propose that they are mediated by influencing cellular adhesion and migration via various signaling pathways and by directly inactivating key cell adhesion surface receptor sites. The evidence for this proposition is reviewed (by summarizing the natural metabolites and their effects influencing cellular adhesion and migration), along with the classical measuring techniques used to gain such evidence. We systematize existing knowledge concerning the mechanisms of how natural metabolites affect adhesion and movement, and their role in gene expression as well. We conclude by highlighting the possibilities to screen natural compounds faster and more easily by applying new label-free methods, which also enable a far greater degree of quantification than the conventional methods used hitherto. We have systematically classified recent studies regarding the effects of natural compounds on cellular adhesion and movement, characterizing the active substances according to their organismal origin (plants, animals or fungi). Finally, we also summarize the results of recent studies and experiments on SARS-CoV-2 treatments by natural extracts affecting mainly the adhesion and entry of the virus.

The fungus Kalmusia longispora is able to cause vascular necrosis on Vitis vinifera.

Fungal diseases in agronomically important plants such as grapevines result in significantly reduced production, pecuniary losses, and increased use of environmentally damaging chemicals. Beside the well-known diseases, there is an increased interest in wood-colonizing fungal pathogens that infect the woody tissues of grapevines. In 2015, a traditional isolation method was performed on grapevine trunks showing symptoms of trunk diseases in Hungary. One isolate (T15142) was identified as Kalmusia longispora (formerly Dendrothyrium longisporum) according to morphological and phylogenetic analyses. To evaluate the pathogenicity of this fungus on grapevines, artificial infections were carried out under greenhouse and field conditions, including the CBS 824.84 and ex-type CBS 582.83 strains. All isolates could be re-isolated from inoculated plants; however, varying virulence was observed among them in terms of the vascular necrosis caused. The incidence and severity of this symptom seemed to be congruent with the laccase-producing capabilities of the isolates. This is the first report on the ability of Kalmusia longispora to cause symptoms on grapevines, and on its possible dependence on laccase secretion.

What is the role of the nitrate reductase (euknr) gene in fungi that live in nitrate-free environments? A targeted gene knock-out study in Ampelomyces mycoparasites.

Mycoparasitic fungi can be utilized as biocontrol agents (BCAs) of many plant pathogens. Deciphering the molecular mechanisms of mycoparasitism may improve biocontrol efficiency. This work reports the first functional genetic studies in Ampelomyces, widespread mycoparasites and BCAs of powdery mildew fungi, and a molecular genetic toolbox for future works. The nitrate reductase (euknr) gene was targeted to reveal the biological function of nitrate assimilation in Ampelomyces. These mycoparasites live in an apparently nitrate-free environment, i.e. inside the hyphae of powdery mildew fungi that lack any nitrate uptake and assimilation system. Homologous recombination-based gene knock-out (KO) was applied to eliminate the euknr gene using Agrobacterium tumefaciens-mediated transformation. Efficient KO of euknr was confirmed by PCR, and visible phenotype caused by loss of euknr was detected on media with different nitrogen sources. Mycoparasitic ability was not affected by knocking out euknr as a tested transformant readily parasitized Blumeria graminis and Podosphaera xanthii colonies on barley and cucumber, respectively, and the rate of mycoparasitism did not differ from the wild type. These results indicate that euknr is not involved in mycoparasitism. Dissimilatory processes, involvement in nitric oxide metabolism, or other, yet undiscovered processes may explain why a functional euknr is maintained in Ampelomyces.

Succession and potential role of bacterial communities during Pleurotus ostreatus production.

There is an increasing interest in studying bacterial-fungal interactions (BFIs), also the interactions of Pleurotus ostreatus, a model white-rot fungus and important cultivated mushroom. In Europe, P. ostreatus is produced on a wheat straw-based substrate with a characteristic bacterial community, where P. ostreatus is exposed to the microbiome during substrate colonisation. This study investigated how the bacterial community structure was affected by the introduction of P. ostreatus into the mature substrate. Based on the results obtained, the effect of the presence and absence of this microbiome on P. ostreatus production in an experimental cultivation setup was determined. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) and amplicon sequencing revealed a definite succession of the microbiome during substrate colonisation and fruiting body production: a sharp decrease in relative abundance of Thermus spp. and Actinobacteria, and the increasing dominance of Bacillales and Halomonas spp. The introduced experimental cultivation setup proved the protective role of the microbial community against competing fungi without affecting P. ostreatus growth. We could also demonstrate that this effect could be attributed to both living microbes and their secreted metabolites. These findings highlight the importance of bacterial-fungal interactions during mushroom production.

The grass root endophytic fungus Flavomyces fulophazii: An abundant source of tetramic acid and chlorinated azaphilone derivatives.

Fungal endophytes are remarkable sources of biologically active metabolites of ecological and pharmacological significance. In this study, fungal isolates producing yellow pigments and originating from grass roots, were identified as the recently described grass root colonizing dark septate endophyte (DSE), Flavomyces fulophazii (Periconiaceae, Pleosporales). While analyzing the metabolite composition of 17 isolates of this fungus, 11 previously undescribed compounds, including four tetramic acids (dihydroxyvermelhotin, hydroxyvermelhotin, methoxyvermelhotin, oxovermelhotin), and seven chlorinated azaphilones (flavochlorines A-G), together with the known tetramic acid vermelhotin, were tentatively identified by high performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS). Among them, flavochlorine A, flavochlorine G, hydroxyvermelhotin and vermelhotin could be isolated by preparative HPLC, thus their structures were also confirmed by nuclear magnetic resonance (NMR) spectroscopy. Vermelhotin was found to be the main compound, reaching its maximum level of 5.5 mg/g in the in vitro cultures of a selected F. fulophazii isolate. A significant amount of vermelhotin was isolated by preparative HPLC from these cultures (4.8 mg from 1.0 g lyophilized culture), confirming the practical utility of F. fulophazii in high-yield vermelhotin production. The main compounds of this endophyte expressed no activity in standardized plant bioassays (i.e., in the Lactuca sativa seed germination and Lemna minor growth tests). An antiproliferative study of the isolated compounds confirmed moderate activity of vermelhotin against a panel of twelve cancer cell lines, with IC50 ranges of 10.1-37.0 µM, without inhibiting the non-cancer Vero cells, suggesting its selectivity towards cancers.

Above-ground parts of white grapevine Vitis vinifera cv. Furmint share core members of the fungal microbiome.

Grapevine (Vitis vinifera) is a reservoir of fungal endophytes that may affect its growth, health status and grape production. Although there is growing interest in comparing fungal communities of mainly red grape varieties across various factors using only high-throughput sequencing, the small-scale mycobiome variations in geographically close vineyards need further examination. We aimed to characterize the fungal microbiome of the above-ground tissues of V. vinifera cv. Furmint in different plant parts, seasons and sites using culture-dependent and culture-independent methods, and in planta fluorescent microscopic visualization techniques. Samples were collected from four sites of the Tokaj wine region in Mád and two reference sites in Eger, Hungary, across different seasons for 2 years. Fungal endophytes of young and mature leaves, flowers and grape bunches were collected at different phenological stages. Based on each technique, Aureobasidium pullulans, Cladosporium spp. and the complex species Alternaria alternata dominated the community at every site, season and plant organ. We found no significant difference among communities in distinct neighbouring vineyards, nor when compared with the distant reference sites. We can conclude that the different shoot parts of the Furmint grapevines harbour a common core group of fungal community in these regions.

Mycology: Rediscovery of a lost model fungus highlights the origin of mycorrhizal symbioses.

Arbuscular mycorrhizae (AM) are the most frequent symbioses of land plants. By reisolating a long-lost fungus from nature, a new study cracks the genomics of an enigmatic fungal-cyanobacterial partnership and reestablishes a valuable model for understanding the AM symbiosis.

Salt Stress Tolerance of Dark Septate Endophytes Is Independent of Melanin Accumulation.

Dark septate endophytes (DSEs) represent a diverse group of root-endophytic fungi that have been isolated from plant roots in many different natural and anthropogenic ecosystems. Melanin is widespread in eukaryotic organisms and possesses various functions such as protecting human skin from UV radiation, affecting the virulence of pathogens, and playing a role in development and physiology of insects. Melanin is a distinctive feature of the cell walls of DSEs and has been thought to protect these fungi from abiotic stress. Melanin in DSEs is assumed to be synthesized via the 1,8-dihydroxynaphthalene (DHN) pathway. Its function in alleviation of salt stress is not yet known. The aims of this study were: (i) investigating the growth responses of three DSEs (Periconia macrospinosa, Cadophora sp., and Leptodontidium sp.) to salt stress, (ii) analyzing melanin production under salt stress and, (iii) testing the role of melanin in salt stress tolerance of DSEs. The study shows that the three DSE species can tolerate high salt concentrations. Melanin content increased in the hyphae of all DSEs at 100 mM salt, but decreased at 500 mM. This was not reflected in the RNA accumulation of the gene encoding scytalone dehydratase which is involved in melanin biosynthesis. The application of tricyclazole, a DHN-melanin biosynthesis inhibitor, did not affect either salt stress tolerance or the accumulation of sodium in the hyphae. In addition, melanin biosynthesis mutants of Leptodontidium sp. did not show decreased growth performance compared to the wild-type, especially not at high salt concentrations. This indicates that DSEs can live under salt stress and withstand these conditions regardless of melanin accumulation.

The new truffle genus Babosia and a new species of Stouffera from semiarid grasslands of Hungary.

Truffles with distinct morphological and anatomical features were collected during a study of hypogeous fungi of semiarid sandy grasslands of the Great Hungarian Plain in Hungary, representing the westernmost localities of the Eurasian steppe belt. None of the ascomata were collected near ectomycorrhizal plant species, and none were identified as ectomycorrhizal during previous surveys in the collection area. We studied morphoanatomical characteristics of these truffles with light and scanning electron microscopy and investigated their phylogenetic positions based on analyses of different nuclear loci. The truffles were found to represent two novel lineages that grouped with the Marcelleina-Peziza gerardii clade of the Pezizaceae. One formed a distinct lineage, for which we propose a new genus Babosia with a new species Babosia variospora characterized by diverse spore ornamentation varying even within one ascus. The truffles in the other lineage clustered with the rarely collected American truffle Stouffera longii and share with it similar spore ornamentation and habitat features. However, our material differs from S. longii by geographic origin, the quick and strong coloration of the ascomata to dark gray at cut surface or bruised area, varying spore number in asci, and smaller spore size; thus, we describe it as a new species, Stouffera gilkeyae.