Diversity of colacosome-interacting mycoparasites expands the understanding of the evolution and ecology of Microbotryomycetes.

Mycoparasites in Basidiomycota comprise a diverse group of fungi, both morphologically and phylogenetically. They interact with their hosts through either fusion-interaction or colacosome-interaction. Colacosomes are subcellular structures formed by the mycoparasite at the host-parasite interface, which penetrate the parasite and host cell walls. Previously, these structures were detected in 19 fungal species, usually by means of transmission electron microscopy. Most colacosome-forming species have been assigned to Microbotryomycetes (Pucciniomycotina, Basidiomycota), a highly diverse class, comprising saprobic yeasts, mycoparasites, and phytoparasites. In general, these myco- and phytoparasites are dimorphic organisms, with a parasitic filamentous morph and saprobic yeast morph. We investigated colacosome-forming mycoparasites based on fungarium material, freshly collected specimens, and cultures of yeast morphs. We characterised the micromorphology of filamentous morphs, the physiological characteristics of yeast morphs, and inferred phylogenetic relationships based on DNA sequence data from seven loci. We outline and employ an epifluorescence-based microscopic method to assess the presence and organisation of colacosomes. We describe five new species in the genus Colacogloea, the novel dimorphic mycoparasite Mycogloiocolax gerardii, and provide the first report of a sexual, mycoparasitic morph in Colacogloea philyla and in the genus Slooffia. We detected colacosomes in eight fungal species, which brings the total number of known colacosome-forming fungi to 27. Finally, we revealed three distinct types of colacosome organisation in Microbotryomycetes. Taxonomic novelties and typifications: New family: Mycogloiocolacaeae Schoutteten & Yurkov; New genus: Mycogloiocolax Schoutteten & Rödel; New species: Colacogloea bettinae Schoutteten & Begerow, C. biconidiata Schoutteten, C. fennica Schoutteten & Miettinen, C. microspora Schoutteten, C. universitatis-gandavensis Schoutteten & Verbeken, Mycogloiocolax gerardii Schoutteten & Rödel; New combinations: Slooffia micra (Bourdot & Galzin) Schoutteten, Fellozyma cerberi (A.M. Yurkov et al.) Schoutteten & Yurkov, Fellozyma telluris (A.M. Yurkov et al.) Schoutteten & Yurkov; Epitypifications (basionyms): Achroomyces insignis Hauerslev, Platygloea micra Bourdot & Galzin, Platygloea peniophorae Bourdot & Galzin; Lectotypification (basionym): Platygloea peniophorae Bourdot & Galzin Citation: Schoutteten N, Yurkov A, Leroux O, Haelewaters D, Van Der Straeten D, Miettinen O, Boekhout T, Begerow D, Verbeken A (2023). Diversity of colacosome-interacting mycoparasites expands the understanding of the evolution and ecology of Microbotryomycetes. Studies in Mycology 106: 41-94. doi: 10.3114/sim.2022.106.02.

Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis.

BACKGROUND: Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host. RESULTS: We have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized. The Agrobacterium transfer DNA (T-DNA) showed predominantly single-copy chromosomal integrations in R. toruloides, which were trackable by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). To demonstrate the application of random T-DNA insertions for strain improvement and gene hunting, 3 T-DNA insertional libraries were screened against cerulenin, nile red and tetrazolium violet respectively, resulting in the identification of 22 mutants with obvious phenotypes in fatty acid or lipid metabolism. Similarly, 5 carotenoid biosynthetic mutants were obtained through visual screening of the transformants. To further validate the gene tagging strategy, one of the carotenoid production mutants, RAM5, was analyzed in detail. The mutant had a T-DNA inserted at the putative phytoene desaturase gene CAR1. Deletion of CAR1 by homologous recombination led to a phenotype similar to RAM5 and it could be genetically complemented by re-introduction of the wild-type CAR1 genome sequence. CONCLUSIONS: T-DNA insertional mutagenesis is an efficient forward genetic tool for gene discovery in R. toruloides and related oleaginous yeast species. It is also valuable for metabolic engineering in these hosts. Further analysis of the 27 mutants identified in this study should augment our knowledge of the lipid and carotenoid biosynthesis, which may be exploited for oil and isoprenoid metabolic engineering.

Deconstructing the evolutionary complexity between rust fungi (Pucciniales) and their plant hosts.

The rust fungi (Pucciniales) are the most speciose natural group of plant pathogens, members of which possess the most complex lifecycles in Fungi. How natural selection works on the Pucciniales has been the subject of several hypotheses in mycology. This study uses molecular age estimation using sequence data from multiple loci, and cophylogeny reconciliation analyses to test hypotheses regarding how the aecial and telial stages in the lifecycle of rust fungi may have differentially impacted their diversification. Molecular age estimates show that the timing of diversification in the Pucciniales correlates with the diversification of their gymnosperm and angiosperm hosts. Host reconciliation analyses suggest that systematic relationships of hosts from the aecial stage of the Pucciniales lifecycle better reflect the systematic relationships among the Pucciniales. The results demonstrate the relative importance of this stage on the overall evolution of the Pucciniales and supports hypotheses made by Leppik over half a century ago. This study represents the first evaluation of how different life stages in the Pucciniales shape the evolution of these fungi.

Phylogeny of yeasts and related filamentous fungi within Pucciniomycotina determined from multigene sequence analyses.

In addition to rusts, the subphylum Pucciniomycotina (Basidiomycota) includes a large number of unicellular or dimorphic fungi which are usually studied as yeasts. Ribosomal DNA sequence analyses have shown that the current taxonomic system of the pucciniomycetous yeasts which is based on phenotypic criteria is not concordant with the molecular phylogeny and many genera are polyphyletic. Here we inferred the molecular phylogeny of 184 pucciniomycetous yeast species and related filamentous fungi using maximum likelihood, maximum parsimony and Bayesian inference analyses based on the sequences of seven genes, including the small subunit ribosomal DNA (rDNA), the large subunit rDNA D1/D2 domains, the internal transcribed spacer regions (ITS 1 and 2) of rDNA including the 5.8S rDNA gene; the nuclear protein-coding genes of the two subunits of DNA polymerase II (RPB1 and RPB2) and the translation elongation factor 1-α (TEF1); and the mitochondrial gene cytochrome b (CYTB). A total of 33 monophyletic clades and 18 single species lineages were recognised among the pucciniomycetous yeasts employed, which belonged to four major lineages corresponding to Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes and Mixiomycetes. These lineages remained independent from the classes Atractiellomycetes, Classiculomycetes, Pucciniomycetes and Tritirachiomycetes formed by filamentous taxa in Pucciniomycotina. An updated taxonomic system of pucciniomycetous yeasts implementing the 'One fungus = One name' principle will be proposed based on the phylogenetic framework presented here.

Phylogenetic classification of yeasts and related taxa within Pucciniomycotina.

Most small genera containing yeast species in the Pucciniomycotina (Basidiomycota, Fungi) are monophyletic, whereas larger genera including Bensingtonia, Rhodosporidium, Rhodotorula, Sporidiobolus and Sporobolomyces are polyphyletic. With the implementation of the "One Fungus = One Name" nomenclatural principle these polyphyletic genera were revised. Nine genera, namely Bannoa, Cystobasidiopsis, Colacogloea, Kondoa, Erythrobasidium, Rhodotorula, Sporobolomyces, Sakaguchia and Sterigmatomyces, were emended to include anamorphic and teleomorphic species based on the results obtained by a multi-gene phylogenetic analysis, phylogenetic network analyses, branch length-based methods, as well as morphological, physiological and biochemical comparisons. A new class Spiculogloeomycetes is proposed to accommodate the order Spiculogloeales. The new families Buckleyzymaceae with Buckleyzyma gen. nov., Chrysozymaceae with Chrysozyma gen. nov., Microsporomycetaceae with Microsporomyces gen. nov., Ruineniaceae with Ruinenia gen. nov., Symmetrosporaceae with Symmetrospora gen. nov., Colacogloeaceae and Sakaguchiaceae are proposed. The new genera Bannozyma, Buckleyzyma, Fellozyma, Hamamotoa, Hasegawazyma, Jianyunia, Rhodosporidiobolus, Oberwinklerozyma, Phenoliferia, Pseudobensingtonia, Pseudohyphozyma, Sampaiozyma, Slooffia, Spencerozyma, Trigonosporomyces, Udeniozyma, Vonarxula, Yamadamyces and Yunzhangia are proposed to accommodate species segregated from the genera Bensingtonia, Rhodosporidium, Rhodotorula, Sporidiobolus and Sporobolomyces. Ballistosporomyces is emended and reintroduced to include three Sporobolomyces species of the sasicola clade. A total of 111 new combinations are proposed in this study.

Insights into Diversity, Distribution, and Systematics of Rust Genus Puccinia.

Puccinia, which comprises 4000 species, is the largest genus of rust fungi and one of the destructive plant pathogenic rust genera that are reported to infect both agricultural and nonagricultural plants with severe illnesses. The presence of bi-celled teliospores is one of the major features of these rust fungi that differentiated them from Uromyces, which is another largest genus of rust fungi. In the present study, an overview of the current knowledge on the general taxonomy and ecology of the rust genus Puccinia is presented. The status of the molecular identification of this genus along with updated species numbers and their current statuses in the 21st century are also presented, in addition to their threats to both agricultural and nonagricultural plants. Furthermore, a phylogenetic analysis based on ITS and LSU DNA sequence data available in GenBank and the published literature was performed to examine the intergeneric relationships of Puccinia. The obtained results revealed the worldwide distribution of Puccinia. Compared with other nations, a reasonable increase in research publications over the current century was demonstrated in Asian countries. The plant families Asteraceae and Poaceae were observed as the most infected in the 21st century. The phylogenetic studies of the LSU and ITS sequence data revealed the polyphyletic nature of Puccinia. In addition, the presences of too short, too lengthy, and incomplete sequences in the NCBI database demonstrate the need for extensive DNA-based analyses for a better understanding of the taxonomic placement of Puccinia.

A Global Overview of Diversity and Phylogeny of the Rust Genus Uromyces.

Uromyces is the second-largest plant pathogenic rust genus, is responsible for numerous diseases, and has major effects on both agricultural and non-agricultural plants. The genus is generally characterized by its unicellular teliospores that help to characterize it and distinguish it from another important rust genus, Puccinia. In this study, a global overview of the diversity and distribution of Uromyces is presented based on both online and offline resources. The information obtained was analyzed for numerical and graphical summaries to provide the diversity and distribution of the genus by country and continent. Besides this, broad taxonomical aspects, a brief life cycle, and other comparative aspects on diversity and distribution were also provided. In addition, a phylogenetic analysis based on the ITS and nLSU DNA sequence data available in GenBank and published literature was performed to examine the intergeneric relationships of Uromyces. The results obtained revealed that the rust genus is found distributed over 150 countries, territories, and occupancies of the world on around 647 plant genera belonging to 95 plant families. Phylogenetic studies based on LSU and ITS sequence data revealed that Uromyces species are polyphyletic and require more DNA-based analyses for a better understanding of their taxonomic placement.

Austropuccinia psidii, causing myrtle rust, has a gigabase-sized genome shaped by transposable elements.

Austropuccinia psidii, originating in South America, is a globally invasive fungal plant pathogen that causes rust disease on Myrtaceae. Several biotypes are recognized, with the most widely distributed pandemic biotype spreading throughout the Asia-Pacific and Oceania regions over the last decade. Austropuccinia psidii has a broad host range with more than 480 myrtaceous species. Since first detected in Australia in 2010, the pathogen has caused the near extinction of at least three species and negatively affected commercial production of several Myrtaceae. To enable molecular and evolutionary studies into A. psidii pathogenicity, we assembled a highly contiguous genome for the pandemic biotype. With an estimated haploid genome size of just over 1 Gb (gigabases), it is the largest assembled fungal genome to date. The genome has undergone massive expansion via distinct transposable element (TE) bursts. Over 90% of the genome is covered by TEs predominantly belonging to the Gypsy superfamily. These TE bursts have likely been followed by deamination events of methylated cytosines to silence the repetitive elements. This in turn led to the depletion of CpG sites in TEs and a very low overall GC content of 33.8%. Compared to other Pucciniales, the intergenic distances are increased by an order of magnitude indicating a general insertion of TEs between genes. Overall, we show how TEs shaped the genome evolution of A. psidii and provide a greatly needed resource for strategic approaches to combat disease spread.

Taxonomic revision of species of Kuehneola and Phragmidium on Rosa, including two new species from China.

Kuehneola japonica and K. warburgiana are the only species of genus Kuehneola that parasitize plants belonging to genus Rosa (Rosaceae). Systematic revision of the genus Phragmidium and related genera by molecular phylogenetic analyses using nuc rDNA internal transcribed spacer (5.8S-ITS2 = ITS2) and nuc rDNA 28S (28S) sequences indicated that K. japonica and K. warburgiana belong to a group of Phragmidium species that also occur on Rosa. Morphological and molecular phylogenetic analyses revealed that these Kuehneola species were transferred to Phragmidium and renamed as P. japonicum and P. warburgianum. Two new Phragmidium species were also discovered on Rosa from China that could be distinguished from other species in the genus based on aeciospore or urediniospore morphology and phylogenetic placement. The first species, P. jiangxiense, is characterized by a urediniospore surface structure with stout spines that are basally embedded in the wrinkled spore wall. The second species, P. leucoaecium, is characterized by an aeciospore surface structure with irregularly elongated verrucae. Taxonomic descriptions and illustrations are provided.

New species of Bannoa described from the tropics and the first report of the genus in South America.

The genus Bannoa consists of four described species associated with dead leaves in southwestern Japan. In this study, we describe three new species, Bannoa guamensis, B. rosea, and B. tropicalis, from the South Pacific island of Guam and Guyana in South America. Isolates were obtained from surfaces of diseased and healthy leaves of plants in the Euphorbiaceae, Asteraceae, and Poaceae. DNA sequences from four gene regions, including nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS), D1-D2 domains of nuc 28S rDNA (28S), nuc 18S rDNA (18S), and a portion of tef1, which encodes translation elongation factor 1-alpha, were produced for phylogenetic analysis. Intercompatibility tests were performed, and subsequent development of clamp connections and basidia were documented for B. tropicalis. Potential life history strategies and association with diseased leaves, including rust-infected leaves, were evaluated across the genus. This is the first report of a species of Bannoa from South America.

Classiculasinensis, a new species of basidiomycetous aquatic hyphomycetes from southwest China.

Classiculasinensis, isolated from decaying leaves from Mozigou, Chongqing Municipality, China, is described as a new species. The new species is a member of basidiomycetous aquatic hyphomycetes which represent a small proportion of all aquatic hyphomycetes. This species falls within the genus Classicula (Classiculaceae, Pucciniomycotina) and is closely related to C.fluitans, based on multiple gene sequence analyses. Morphologically, it is characterised by the apical, hyaline, obclavate or navicular conidia with several hair-like lateral appendages and by its holoblastic and monoblastic conidiogenesis, with a flat un-thickened conidiogenous locus. Clamp connections and haustorial branches were often observed in culture.

Dismantling a complex of anther smuts (Microbotryum) on carnivorous plants in the genus Pinguicula.

The anther smuts of the genus Microbotryum are known from host plant species belonging to the Caryophyllaceae, Dipsacaceae, Lamiaceae, Lentibulariaceae, Montiaceae, and Primulaceae. Of these, the anther smuts on Caryophyllaceae, in particular on Silene spp., are best known because they include model organisms studied in many disciplines of fungal biology. For Microbotryum species parasitic on Caryophyllaceae, a high degree of host specificity was revealed and several cryptic species were described. In contrast, the host specificity within Microbotryum pinguiculae occurring in anthers of different Pinguicula species (Lentibulariaceae) has not been investigated in detail until now. The anther smuts on Pinguicula alpina, P. villosa, and P. vulgaris, on which M. pinguiculae was described, were analyzed using nuc rDNA ITS1-5.8S-ITS2 and nuc rDNA 28S D1-D2 sequences and morphology to determine if they belong to one polyphagous species or rather represent three host-specific species. The results of the morphological investigations revealed no decisive differences between the anther smuts on different Pinguicula species. However, genetic divergence and molecular phylogenetic analyses, which split the specimens according to host plant species, supported host specificity of the anther smuts on different Pinguicula species. Accordingly, in addition to Microbotryum pinguiculae s. str. on Pinguicula vulgaris, M. alpinum sp. nov. on P. alpina from Europe and M. liroi sp. nov. on P. villosa from Asia are described and illustrated.

The species of Coleosporium (Pucciniales) on Solidago in North America.

Species of Coleosporium (Pucciniales) are rust fungi that typically alternate between pines and angiosperms. In North America, species of Coleosporium often infect Solidago (goldenrods), although their taxonomy on these hosts is unresolved. Joseph. C. Arthur and George B. Cummins regarded these as a single species, Coleosporium solidaginis (fide Arthur) or C. asterum (fide Cummins), but later inoculation studies demonstrated the presence of more than one species, distinguishable by their aecial hosts. A more recent taxonomic study of Coleosporium found that specimens on Solidago identified as C. asterum in North America were not conspecific with the type, which is from Japan, prompting the present study. Herein, we conducted a systematic study on ca. 60 collections of Coleosporium infecting species of Asteraceae from North America using regions of ribosomal DNA and morphology of teliospores and basidia. Our data indicate at least three species of Coleosporium occur on Solidago in North America, C. solidaginis, C. montanum comb. nov., which is proposed for the taxon that has commonly been identified as C. asterum in North America, and C. delicatulum, all of which can be differentiated by morphology of their basidia. In addition, the challenges of marker selection for molecular barcoding of rust fungi is discussed.

Phylogenetics and Phylogenomics of Rust Fungi.

Rust fungi (Pucciniales) are the most speciose and the most complex group of plant pathogens. Historically, rust taxonomy was largely influenced by host and phenotypic characters, which are potentially plastic. Molecular systematic studies suggest that the extant diversity of this group was largely shaped by host jumps and subsequent shifts. However, it has been challenging to reconstruct the evolutionary history for the order, especially at deeper (family-level) nodes. Phylogenomics offer a potentially powerful tool to reconstruct the Pucciniales tree of life, although researchers working at this vanguard still face unprecedented challenges working with nonculturable organisms that possess some of the largest and most repetitive genomes now known in kingdom fungi. In this chapter, we provide an overview of the current status and special challenges of rust genomics, and we highlight how phylogenomics may provide new perspectives and answer long-standing questions regarding the biology of rust fungi.

Transmission electron microscopy of Tuberculina species (Helicobasidiales) reveals an unique mode of conidiogenesis within Basidiomycota.

Tuberculina species represent the asexual life stage of the plant-parasitic sexual genus Helicobasidium. Tuberculina species are distributed all over the world, living in antagonistic symbiosis with over 150 rust species from at least 15 genera. Within the Basidiomycota, besides the spermogonia of rust fungi, only Tuberculina species develop distinct fructifications in the haplophase. However, the knowledge of conidiogenesis in Tuberculina is meagre. Therefore, conidial development in Tuberculina maxima, Tuberculina persicina, and Tuberculina sbrozzii was studied using transmission electron microscopy, and compared to each other as well as to spermatia formation in rust fungi. Significant ultrastructural characteristics such as the movement of nuclei in the process of conidium formation, and formation of the initial and late stages of conidiogenesis are documented. The mode of conidiogenesis of Tuberculina species is unique within the Basidiomycota in that (1) it is realized by haploid fructifications, (2) it is holoblastic, without annellidic proliferation, (3) the nucleus of the conidiogenous cell moves towards the forming conidium, divides, and no daughter nucleus remains inside the conidiogenous cell, and (4) the conidiogenous cell retains only cytoplasmic residues after the development of a single conidium, and a successive conidium is not produced.

In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections.

BACKGROUND: Rust fungi are obligate pathogens with multiple life stages often including different spore types and multiple plant hosts. While individual rust pathogens are often associated with specific plants, a wide range of plant species are infected with rust fungi. To study the interactions between these important pathogenic fungi and their host plants, one must be able to differentiate fungal tissue from plant tissue. This can be accomplished using the In situ hybridization (ISH) protocol described here. RESULTS: To validate reproducibility using the ISH protocol, samples of Chrysanthemum × morifolium infected with Puccinia horiana, Gladiolus × hortulanus infected with Uromyces transversalis and Glycine max infected with Phakopsora pachyrhizi were tested alongside uninfected leaf tissue samples. The results of these tests show that this technique clearly distinguishes between rust pathogens and their respective host plant tissues. CONCLUSIONS: This ISH protocol is applicable to rust fungi and potentially other plant pathogenic fungi as well. It has been shown here that this protocol can be applied to pathogens from different genera of rust fungi with no background staining of plant tissue. We encourage the use of this protocol for the study of plant pathogenic fungi in paraffin embedded sections of host plant tissue.

Uromyces hawksworthii nom. nov. for Aecidium goyazense, on Phthirusa stelis (Loranthaceae) from the Brazilian Cerrado.

The sexual morph of Aecidium goyazense collected in the Brazilian Cerrado was morphologically characterized by light microscopy and SEM, and shown to be a species of Uromyces, for which the name Uromyces hawksworthii nom. nov. is introduced, and designated as its epitype. This is the second Uromyces species known to infect the tropical genus Phthirusa (Loranthaceae). DNA sequences were generated from the ITS and 28S rRNA (LSU) regions of DNA recovered from aeciospores as well as teliospores. This fungus is compared with other Uromyces species known from Loranthaceae.