Re-examination of the Southern Hemisphere truffle genus Amylascus (Pezizaceae, Ascomycota) and characterization of the sister genus Nothoamylascus gen. nov.

Amylascus is a genus of ectomycorrhizal truffles within Pezizaceae that is known from Australia and contains only two described species, A. herbertianus and A. tasmanicus. Species of Amylascus are closely related to truffles (Pachyphlodes, Luteoamylascus) and cup fungi (Plicariella) from the Northern Hemisphere. Here we reevaluate the species diversity of Amylascus and related taxa from southern South America and Australia based on new morphological and molecular data. We identify previously undocumented diversity and morphological variability in ascospore color, ascospore ornamentation, hymenial construction, epithecium structure and the amyloid reaction of the ascus in Melzer's reagent. We redescribe two Amylascus species from Australia and describe seven new Amylascus species, five from South America and two from Australia. This is the first report of Amylascus species from South America. We also describe the new South American genus Nothoamylascus as sister lineage to the Pachyphlodes-Amylascus-Luteoamylascus clade (including Amylascus, Luteoamylascus, Pachyphlodes, and Plicariella). We obtained ITS sequences of mitotic spore mats from Nothoamylascus erubescens gen. & sp. nov. and four of the seven newly described Amylascus species, providing the first evidence of mitotic spore mats in Amylascus. Additional ITS sequences from mitotic spore mats reveal the presence of nine additional undescribed Amylascus and one Nothoamylascus species that do not correspond to any sampled ascomata. We also identify three additional undescribed Amylascus species based on environmental sequences from the feces of two grounddwelling bird species from Chile, Scelorchilus rubecula and Pteroptochos tarnii. Our results indicate that ascomata from Amylascus and Nothoamylascus species are rarely collected, but molecular data from ectomycorrhizal roots and mitotic spore mats indicate that these species are probably common and widespread in southern South America. Finally, we present a time-calibrated phylogeny that is consistent with a late Gondwanan distribution. The time since the most recent common ancestor of: 1) the family Pezizaceae had a mean of 276 Ma (217-337 HPD); 2) the Amylascus-Pachyphlodes-Nothoamylascus-Luteoamylascus clade had a mean of 79 Ma (60-100 HPD); and 3) the Amylascus-Pachyphlodes clade had a mean of 50 Ma (38-62 HPD). The crown age of Pachyphlodes had a mean of 39 Ma (25-42 HPD) and Amylascus had a mean age of 28 Ma (20-37 HPD), falling near the Eocene-Oligocene boundary and the onset of the Antarctic glaciation (c. 35 Ma). Citation: Healy RA, Truong C, Castellano MA, et al. 2023. Re-examination of the Southern Hemisphere truffle genus Amylascus (Pezizaceae, Ascomycota) and characterization of the sister genus Nothoamylascus gen. nov. Persoonia 51: 125-151. doi: 10.3767/persoonia.2023.51.03.

Eight new Elaphomyces species (Elaphomycetaceae, Eurotiales, Ascomycota) from eastern North America.

The hypogeous, sequestrate ascomycete genus Elaphomyces is one of the oldest known truffle-like genera. Elaphomyces has a long history of consumption by animals in Europe and was formally described by Nees von Esenbeck in 1820 from Europe. Until recently most Elaphomyces specimens in North America were assigned names of European taxa due to lack of specialists working on this group and difficulty of using pre-modern species descriptions. It has recently been discovered that North America has a rich diversity of Elaphomyces species far beyond the four Elaphomyces species described from North America prior to 2012. We describe eight new Elaphomyces species (E. dalemurphyi, E. dunlapii, E. holtsii, E. lougehrigii, E. miketroutii, E. roodyi, E. stevemilleri and E. wazhazhensis) of eastern North America that were collected in habitats from Quebec, Canada south to Florida, USA, west to Texas and Iowa. The ranges of these species vary and with continued sampling may prove to be larger than we have established. Castellano has studied authentic material of all European Elaphomyces species published through 2016 and it is interesting to note that many Elaphomyces species from eastern North America have morphological similarities but with distinct morphological differences to a number of European Elaphomyces species. Citation: Castellano MA, Crabtree CD, Mitchell D, Healy RA (2020). Eight new Elaphomyces species (Elaphomycetaceae, Eurotiales, Ascomycota) from eastern North America. Fungal Systematics and Evolution 7: 113-131. doi: 10.3114/fuse.2021.07.06.

Three independent evolutionary events of sequestrate Lactifluus species in Australasia.

Three Australian species with sequestrate basidiome forms are recorded for the first time in the genus Lactifluus based on nuclear ITS-LSU and morphological data. These species represent three rare independent evolutionary events resulting in sequestrate basidiomes arising from agaricoid species in three different sections in two subgenera. All three species have highly reduced basidiome forms, and no species with intermediate forms have been found. Lactifluus dendriticus is unique in the genus in having highly branched, dendritic terminal elements in the pileipellis. We provide full descriptions of two species: Zelleromyces dendriticus (= Lactifluus dendriticus comb. nov.) in Lactifluus subg. Lactifluus sect. Gerardii, and Lactifluus geoprofluens sp. nov. in Lf. subg. Lactifluus sect. Lactifluus. A reduced description is provided for the third, Lactifluus sp. prov. KV181 in Lf. subg. Pseudogymnocarpi sect. Pseudogymnocarpi, as it is currently known from a single sequence.

Hysterangiales revisited: expanded phylogeny reveals new genera and two new suborders.

Hysterangiales (Phallomycetidae, Agaricomycetes, Basidiomycota) is a diverse, nearly cosmopolitan order of predominantly hypogeous, sequestrate, ectomycorrhizal fungi. Expanding on previously published phylogenies, we significantly increased sampling of Hysterangiales specimens, emphasizing representatives from Australia. Using protein-coding genes atp6 (adenosine triphosphate synthase subunit 6) and tef1 (translation elongation factor 1-á), we recovered 26 provisional novel genera, and corroborated existing genera and families. Further, two new suborders (Phallogastrineae subord. nov. and Hysterangineae subord. nov.) and a new family (Phallogastraceae fam. nov.) are described, and three new combinations made to Phallogaster. Aspects of classification and biogeography are presented.

Balsamia (Sequestrate Helvellaceae, Ascomycota) in western North America.

Balsamia, a hypogeous, sequestrate genus in the Helvellaceae, has been characterized variously as having three to eight species in North America, and these have been considered either different from or conspecific with European species. No available modern systematic treatment of Balsamia exists to allow for accurate identification at the species level. We sequenced DNA from recent western North American Balsamia collections, assessed relationships by sequence similarity, and identified molecular taxonomic units. From these data, we determined which matched descriptions and types of named species. ITS sequences supported 12 Balsamia species in western North America, five originally described by Harkness and Fischer and seven new species that we describe here. No sequences from Balsamia collections in western North America were nested among those of European species. We found no clear evidence for separation of Balsamia into multiple genera.

Three new black Elaphomyces species (Elaphomycetaceae, Eurotiales, Ascomycota) from eastern North America with notes on selected European species.

We describe three new species of Elaphomyces from eastern North America. Of the three, Elaphomyces loebiae is the rarest, known only from North Carolina and South Carolina, and appears to associate primarily with ectomycorrhizal hardwoods but possibly also with conifers. Elaphomyces cibulae is widely distributed but disjunct from Florida, Mississippi, and North Carolina. Elaphomyces cibulae seems to primarily associate with Quercus species. Elaphomyces mitchelliae has the widest distribution of the three species, from Florida, Maryland, North Carolina, Virginia, and West Virginia, and appears to associate with either ectomycorrhizal hardwoods and/or conifers. In the course of comparing our new Elaphomyces species to previously described European species we discovered that E. persoonii var. minor is conspecific in all essential details with and thus a synonym of E. cyanosporus.

Subcellular localization and immunodetection of movement proteins of olive latent virus 1.

Polyclonal sera raised to Escherichia coli-expressed movement proteins encoded by ORF 3 (p8K) and ORF 4 (p6K) of olive latent virus 1, were used for their immunodetection in infected Nicotiana benthamiana plants. In subfractionated locally infected tissues 4 days post inoculation (d.p.i.) that were analysed by Western blot, p8K was found in the fast-sedimenting fractions P1 and P30 containing membranous material and/or cell organelles and, likely, the fibrous structures mentioned below, but not in the soluble protein-containing supernatant. No p6K could be detected in these extracts. In locally inoculated leaves p8K began to accumulate from 2 d.p.i onwards reaching its peak at 4 d.p.i. Intracellular immunogold labelling of cells from locally and systemically infected tissues localized p8K primarily in fibrous inclusions made up of thin filaments with a helical structure present in the cytoplasm of locally and systemically infected cells. In systemic infections a light and scattered labelling was observed in the cytoplasm and near the cell wall. The specific serum to p6K did not label the fibrous structures and failed to recognize its antigen in systemically and locally infected tissues except at 4 d.p.i., when scattered labelling was observed in the cytoplasm and near plasmodesmata.

Opposite effects of low and high doses of arginine on glutamate-induced nitric oxide formation in rat substantia nigra.

L-arginine is a very versatile amino acid that is involved in many important physiological processes such as protein, nitric oxide (NO), agmatine, putrescine, urea, L-ornithine or creatine synthesis and is essential for posttranslational arginylation of protein. The present study was designed to evaluate in vivo the effect of L-arginine on NO production in substantia nigra. In vivo spectroscopic and voltammetric studies were addressed in rats to record modifications in methemoglobin and NO levels under glutamate stimulation. Results showed that, under physiological L-arginine extracellular concentration, the intranigral infusion of glutamate produced an increase in NO levels. When a low dose of L-arginine was co-infused with glutamate, a persistent and higher increase in NO levels was observed. The co-infusion of glutamate with a moderate dose of L-arginine induced drastic and persistent NO production. It was also observed that high doses of either L-arginine or D-arginine inhibit NO production. Subsequently, these data show that L-arginine and D-arginine are involved in a mechanism that inhibits NO production.

Multiple origins of sequestrate fungi related to Cortinarius (Cortinariaceae).

The aim of the present study was to investigate the phylogeny and evolution of sequestrate fungi (with gastroid or partially exposed basidiomes) in relation to their gilled relatives from the Cortinariaceae (Basidiomycetes). Phylogenetic analyses of 151 ITS sequences from 77 gilled species and 37 sequestrate taxa were performed using maximum parsimony and maximum likelihood methods. Results show that sequestrate basidiome forms occur in all three major ectomycorrhizal lineages of Cortinariaceae: the clades Cortinarius, Hebeloma/Hymenogaster/Naucoria, and Descolea. However, these forms do not appear within the saprobic outgroup Gymnopilus, indicating multiple origins of sequestrate forms from ectomycorrhizal ancestors. Additionally, within the Cortinarius clade sequestrate forms have multiple origins: emergent Cortinarius spp., Thaxterogaster, Quadrispora, Protoglossum, and two Hymenogaster spp. (H. remyi, H. sublilacinus) share common ancestors with Cortinarius spp., but these sequestrate genera are not closely related to each other (with exception of Thaxterogaster and Quadrispora). Hymenogaster sensu stricto, Setchelliogaster, and Descomyces were placed in the two other major clades. Thus, sequestrate taxa evolved independently many times within brown-spored Agaricales. Furthermore, emergent, secotioid, and gastroid forms have evolved independently from each other, and so are not necessarily intermediate forms. After their establishment, these apparently morphologically stable taxa show a tendency to radiate.

Immunodetection and subcellular localization of the proteins encoded by ORF 3 of grapevine viruses A and B.

Polyclonal antisera were raised to Escherichia coli-expressed ORF3 products (putative movement proteins) of Grapevine virus A (GVA) and Grapevine virus B (GVB) (genus Vitivirus), and used for their immunodetection in infected plants. Western blot analysis of subfractionated cellular compartments showed that the distribution of both proteins was comparable to that of plant virus movement proteins, as they were transiently present in a crude membrane fraction and accumulated in a cell wall-enriched fraction. The GVA ORF3-encoded protein, but not the comparable GVB protein, was also present in large amount in a cytoplasmic soluble fraction. Intracellular immunogold labelling localized these proteins in the cell wall and plasmodesmata of infected cells and, especially for GVA, in association with cytoplasmic virus aggregates.

Grapevine fleck virus-like viruses in Vitis.

Two sets of degenerate primers for the specific amplification of 572-575 nt and 386 nt segments of the methyltransferase and RNA- dependent RNA polymerase cistrons of members of the genera Tymovirus and Marafivirus and of the unassigned virus Grapevine fleck virus (GFkV) were designed on the basis of available sequences. These primers were used for amplifying and subsequent cloning and sequencing part of the open reading frame 1 of the genome of GFkV, Grapevine asteroid mosaic-associated virus (GAMaV) and of another previously unreported virus, for which the name Grapevine red globe virus (GRGV) is proposed. Computer-assisted analysis of the amplified genome portions showed that the three grapevine viruses are phylogenetically related with one another and with sequenced tymoviruses and marafiviruses. The relationships with tymoviruses was confirmed by the type of ultrastructural modifications induced in the host cells. RdRp-specific degenerate primers were successfully used for the aspecific detection of the three viruses in crude grapevine sap extracts. Specific virus identification was obtained with RT-PCR using antisense virus-specific primers.

Synthesis of infectious transcripts of olive latent virus 1: genes required for RNA replication and virus movement.

Genomic RNA of olive latent virus 1 (OLV-1) contains five open reading frames (ORFs) encoding proteins of 23, 82, 8, 6 and 30 (CP) kDa. A full-length cDNA copy of OLV-1RNA was prepared and cloned in a low-copy-number vector (pMUC-19) downstream of or T7 RNA polymerase promoter. Transcripts derived from this template, denoted pMUC-OLV, were highly infectious when inoculated in local and systemic hosts and infected tissues contained virus-like particles. Genes required for replication and virus movement were mapped by site-directed and deletion mutogenesis of the pMUC-OLV. ORF1 and ORF2 mutants were not viable, suggesting that replication requires the 23 and 82 kDa proteins. The 8 and 6 kDa polypeptides were involved in cell-to-cell movement, since their absence did not interfere with RNA replication but prevented systemic infection of inoculated plants. Mutant clones in R and S domains of the CP gene could replicate, but they did not systemically infect Nicotiana benthamiana, indicating that the CP gene is required for OLV-1 long-distance translocation. Mutant clones with large deletions in the CP gene were not viable, probably due to loss of 3'-proximal sequences required for RNA replication.

Action of antidepressant drugs on maternal stress-induced hypoactivity in female rats.

It has been reported that rats forced to swim in a restricted space assume, after initial frenzied attempts to escape, an immobile posture. Porsolt et al. referred to this phenomenon as "behavioral despair", an animal model of depression. Prenatal stress induces an increase of behavioral depression in adult female offspring. This study presents new evidence supporting the hypothesis that maternal stress during gestation increases the risk of depression in the offspring since immobility time was modified by antidepressant drugs (tricyclics and an atypical antidepressant). In rats, amitriptyline (5 mg/kg), imipramine (5 mg/kg) and nomifensine (1 mg/kg) decreased the immobility time in Porsolt test in offspring of mothers stressed during gestation. Moreover, increasing doses of amitriptyline (1, 5, 25 and 40 mg/kg) reduced depression in the forced swimming test in dose-dependent manner.

Subcellular localization and in vivo identification of the putative movement protein of olive latent virus 2.

The gene encoding the 36.5 kDa ('36K') nonstructural protein located on RNA3 of olive latent virus 2 (OLV-2) was cloned, expressed with the Escherichia coli pGEX-2T system and the purified protein used to raise a polyclonal antiserum. Immunoblot analysis of OLV-2-infected Nicotiana benthamiana plants showed that the 36K protein accumulated in the early stages of infection and was associated with a subcellular fraction enriched in cytoplasmic membranes. In infected cells there were tubular structures, some containing virus-like particles, scattered in the cytoplasm or protruding from or penetrating the cell wall at the plasmodesmata. Immunogold labelling localized the 36K protein in the plasmodesmata of OLV-2-infected cells and showed it to be associated with virus-containing tubules. Leaf trichome cells of N. tabacum plants, transformed with a 36K-green fluorescent protein (GFP) fusion construct, revealed localized fluorescence in the cell walls, possibly due to association of the fusion protein with plasmodesmata. When the same 36K-GFP fusion protein was expressed in N. tabacum protoplasts, long tubular fluorescent structures protruded from the protoplast surface, suggesting that the 36K protein is responsible for tubule induction. The conclusion is drawn that this protein is likely to be the OLV-2 movement protein, mediating cell-to-cell virus movement, and that movement is by a tubule-guided mechanism.

Spontaneous firing of nigrostriatal dopaminergic neurons in split-brain rats.

The regulatory action of the contralateral brain side on nigrostriatal dopaminergic (NSDA) neurons was examined in anesthetized rats using extracellular single-unit recording techniques. The spontaneous activity of NSDA cells was recorded from split-brain, sham-lesioned or intact-brain rats. The three groups of cells did not differ in antidromic latency and firing rate. However, cells from split-brain rats exhibited a more regular pattern of discharge than those recorded in the two control groups as shown by autocorrelograms and the standard deviation and variation coefficient of the interspike intervals. In addition, burst firing was found to be markedly reduced in the split-brain group. It is concluded that decussating pathways are involved in the regulation of the discharge pattern of NSDA neurons.

Transfected D2 short dopamine receptors inhibit voltage-dependent potassium current in neuroblastoma x glioma hybrid (NG108-15) cells.

Two isoforms of the D2 dopamine receptor exist, termed D2 short (D2s) and D2 long, which differ by the presence or absence of 29 amino acids. To examine the possible coupling of the D2s isoform to voltage-dependent K+ current, NG108-15 cells that were transfected with and stably express this isoform were studied using whole-cell patch-clamp techniques. In transfected, but not untransfected, cells dopamine and quinpirole (QUIN) reduced the normally observed peak outward K+ current, and this effect was abolished by the D2 antagonist sulpiride but not by the alpha 2-adrenergic receptor antagonist idazoxan or the D1 antagonist (R)-(+)-SCH-23380. The D1 receptor agonist SKF 38393 had no effect. QUIN-induced inhibition of K+ current was prevented by loading the cells with the Ca(2+)-chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, suggesting a critical role for intracellular Ca2+ mobilization. In contrast, reduction of the concentration of extracellular Ca2+ and inclusion of the Ca2+ channel blocker cobalt did not modify the reduction of K+ current produced by stimulation of D2s receptors. A critical role for intracellular calcium mobilization in the observed effects was further supported by the observation that increases in cytosolic Ca2+ produced by thapsigargin mimicked the effect of QUIN, whereas intracellular ryanodine, which blocks Ca2+ mobilization, abolished the QUIN responsiveness. Finally, the effect of D2S activation on K+ current was not modified by pretreatment of the cells with pertussis toxin. These results suggest that the D2s dopamine receptor expressed in NG108-15 cells inhibits the activity of native K+ current via a mechanism that is dependent upon the mobilization of intracellular Ca2+ and does not involve a pertussis toxin-sensitive G protein.

Properties of a filamentous virus isolated from grapevines affected by corky bark.

A virus with highly flexuous filamentous particles c. 800 nm long, showing distinct transverse striations was isolated with high frequency (60%) by inoculation of Nicotiana occidentalis with sap from grapevine accessions indexing positive for corky bark. The virus, for which the name grapevine virus B (GVB) is proposed, has an ssRNA genome with mol. wt. of c. 2.5 x 10(6) Da (c. 7600 nt) and coat protein subunits with mol. wt. of c 23,000 Da. GVB has a very restricted herbaceous host range and was experimentally transmitted by the mealybug Pseudococcus ficus. The physicochemical and ultrastructural properties of GVB resemble those of closteroviruses. However, it is serologically unrelated to other grapevine closteroviruses including grapevine virus A, with which it shares some biological and physicochemical properties.

Production, characterization and use of monoclonal antibodies to grapevine virus A.

Four stable hybridoma cell lines secreting monoclonal antibodies to grapevine closterovirus A (GVA) were obtained by fusing spleen cells of immunized BALB/c mice with mouse myeloma cell line Sp2/0-Ag 14. In ELISA all MAbs reacted with virus in leaf extracts from Nicotiana benthamiana, glass-house-, field-, or in vitro-grown grapevines, or with cortical shavings from mature grape canes. In IEM tests, only one of the MAbs (PA3.F5) decorated virus particles on the entire surface. This MAb was likely induced by a surface antigenic determinant, whereas the other three MAbs (PA3.D 11, PA3.C 6, and PA3.B 9) were originated by cryptotopes. Coupling polyclonal antibodies for coating protein A-sensitized plates, and monoclonal antibody conjugates for antigen detection, gave highly efficient and reproducible results for identification of GVA in field-grown grapevines.

Properties of a previously undescribed grapevine nepovirus from Tunisia.

A virus with isometric particles c. 30 nm in diameter and angular contour was isolated by inoculation of sap from a Tunisian grapevine with mild mottling and leaf deformation. The virus sedimented in sucrose density gradients as three components: T (empty shells), M (particles containing a molecule of ssRNA with an apparent size of 5,800 nucleotides, constituting 35% of the particle weight) and B (particles containing a molecule of ssRNA with apparent size of 6,800 nucleotides, constituting 41% of the particle weight). Virus particles had buoyant densities of 1.31 (T), 1.45 (M), and 1.49 g/cm3 (B) in cesium chloride equilibrium gradients. The coat protein subunits consisted of a single polypeptide with mol. wt. of c. 59,000 daltons. An antiserum was produced with a titer of 1:256, which did not react with healthy plant antigens. Cells of artificially infected herbaceous hosts showed cytoplasmic vesiculate-vacuolate inclusion bodies, virus-containing tubules, mostly associated with plasmodesmata and/or cell wall protrusions, and crystalline aggregates of virus particles and empty capsids. The physicochemical and ultrastructural properties of this virus resemble very much those of nepoviruses. However, it was serologically unrelated to 19 different members of the group, including all those reported to infect grapevines. Therefore, the virus is possibly a hitherto unreported nepovirus for which the name of grapevine Tunisian ringspot virus (GTRV) is proposed.

Nigrostriatal dopaminergic cell activity is under control by substantia nigra of the contralateral brain side: electrophysiological evidence.

The biochemical balance between right- and left-ascending DA systems is an essential factor to regulate behavioral lateralization. However, there is no electrophysiological evidence for the regulation of interhemispheric DA systems. In the present paper we report electrophysiological evidence supporting the hypothesis that the A9 DA cells are under control of the contralateral substantia nigra. The activity of more than 80% of the A9 cells recorded was affected by contralateral SN stimulation. This is a very high proportion because the previously reported response of A9 cells to ipsilateral caudate stimulation is proportionally lower. The potency of a stimulus, estimated as the number of action potentials induced or inhibited by each electrical stimulation of the contralateral substantia nigra or by the percentage of modification in the number of action potentials induced or inhibited in relation to the spontaneous potential expected, was also high. The response to the contralateral stimulation was complex. Fifty-four percent of all the DA cells studied showed more than a single response. Forty-four percent showed at least one stimulation and at least one inhibition. Because 1) the percentage of cells with at least one stimulation (70%) was higher than the percentage of cells with at least one inhibition (56%), and 2) the potency of stimulations was higher than the inhibition potency, the present data provide evidence that contralateral control of A9 cells is mainly excitatory. The percentage of cells activated by contralateral stimulation was high, between 30 ms and 220 ms and between 400 ms and 700 ms. The probability of inhibition was higher than the probability of activation between 10 ms and 30 ms and between 230 and 380 ms.(ABSTRACT TRUNCATED AT 250 WORDS)