Asexual Fungi Research Articles

Anastomosis groups and molecular variation in Pseudocercospora griseola.

The fungus Pseudocercospora griseola is the causal agent of angular leaf spot, a common bean (Phaseolus vulgaris L.) disease. The successful development of angular leaf spot-resistant cultivars depends on understanding the levels of variability in pathogen populations. P. griseola shows great pathogenic and genetic variation using inoculation on differential bean cultivars and molecular markers. Nevertheless, how this variability develops is not clearly understood. Parasexuality has been presented as a mechanism used by asexual fungi to increase variation. Hyphal fusion is the first step for the parasexual cycle, and it can be considered an additional trait for population studies. The aim of this study was to identify hyphal fusion (anastomosis) among P. grisola isolates and to evaluate the variability of isolates using analyses of anastomosis groups and RAPD markers. Hyphal anastomosis was observed in all isolates. Three isolates showed 85.0% compatibility and were compatible with 17 isolates. This is the first report of the occurrence of anastomosis between P. griseola isolates. Sixteen anastomosis groups were observed and only one group was formed by five isolates (Pg-01, Pg-02, Pg-07, Pg-08, and Pg-12). There was a large number of anastomosis groups and absence of clustering among isolates for hyphal fusion, demonstrating the existence of genetic variability for loci involved in the control of this trait. High genotypic variability by RAPD markers was detected, but there was no relationship between genotype and anastomosis group. More studies are necessary to elucidate further the process of asexual recombination in this phytopathogen.

New record of Porobeltraniella porosa (Piroz. & S.D. Patil) Gusmão, 2004 (Fungi: asexual Ascomycota) for South America

Porobeltraniella porosa (Piroz. & S.D. Patil) Gusmao is an asexual fungus colonizing leaf litter. Previously known for India, from where it was described, and for Australia and Mexico, now it has been collected for the first time in Brazil, in Ribeirao Preto county, Sao Paulo State. The characteristics of the Brazilian material are given here and its geographical distribution is expanded to South America.

Barrage formation is independent from heterokaryon incompatibility in Verticillium dahliae

Barrage formation has been traditionally used for the assessment of mycelial compatibility in many fungi and has often been assumed to represent a non-self recognition phenotype that is directly associated with vegetative incompatibility in these organisms. In this work, the optimal growth conditions for large-scale studies of barrage formation in the asexual fungus Verticillium dahliae were determined, and they were used for the analysis of a diverse collection comprising 69 isolates of V. dahliae and related species. Barrage formation was very frequent on a defined complete agar medium within V. dahliae and between species of the genus. However, it was not correlated with the classification of V. dahliae isolates into Vegetative Compatibility Groups (VCGs) (based on the standard method using complementary nit mutants), as it was recorded at high frequencies both within and between V. dahliae VCGs. The high overall frequency of barrage formation demonstrated the presence of a higher level of mycelial incompatibility in V. dahliae than heterokaryon incompatibility assessed by forcing complementary nit mutants to form heterokaryons under selective conditions. The possible association of barrage formation with morphological characteristics of the fungal colonies was investigated, and a negative correlation of frequency and intensity of barrages with the isolates’ capacity for pigment production was detected.

Recombination between clonal lineages of the asexual fungus Verticillium dahliae detected by genotyping by sequencing.

Most asexual species of fungi have either lost sexuality recently, or they experience recombination by cryptic sexual reproduction. Verticillium dahliae is a plant-pathogenic, ascomycete fungus with no known sexual stage, even though related genera have well-described sexual reproduction. V. dahliae reproduces mitotically and its population structure is highly clonal. However, previously described discrepancies in phylogenetic relationships among clonal lineages may be explained more parsimoniously by recombination than mutation; therefore, we looked for evidence of recombination within and between clonal lineages. Genotyping by sequencing was performed on 141 V. dahliae isolates from diverse geographic and host origins, resulting in 26,748 single-nucleotide polymorphisms (SNPs). We found a strongly clonal population structure with the same lineages as described previously by vegetative compatibility groups (VCGs) and molecular markers. We detected 443 recombination events, evenly distributed throughout the genome. Most recombination events detected were between clonal lineages, with relatively few recombinant haplotypes detected within lineages. The only three isolates with mating type MAT1-1 had recombinant SNP haplotypes; all other isolates had mating type MAT1-2. We found homologs of eight meiosis-specific genes in the V. dahliae genome, all with conserved or partially conserved protein domains. The extent of recombination and molecular signs of sex in (mating-type and meiosis-specific genes) suggest that V. dahliae clonal lineages arose by recombination, even though the current population structure is markedly clonal. Moreover, the detection of new lineages may be evidence that sexual reproduction has occurred recently and may potentially occur under some circumstances. We speculate that the current clonal population structure, despite the sexual origin of lineages, has arisen, in part, as a consequence of agriculture and selection for adaptation to agricultural cropping systems.

Marine fungi of Saudi Arabia

Abstract This study extends our knowledge of the diversity of marine fungi from Saudi Arabia, which is virtually unknown. Thirty-seven fungi (28 ascomycetes, eight asexual fungi, and one basidiomycete) were recorded from 457 samples of driftwood and intertidal decayed wood of Avicennia marina collected from three sites along the Red Sea coast of Saudi Arabia. The three sites include two mangrove sites, namely: Yanbu and Farasan Island, while the third site is a sandy shore nearby Yanbu city. Thirty-one fungi were new records for Saudi Arabia. Swampomyces triseptatus is the only species common to the three sites. The recorded marine fungi were latitudinally distributed with fungi recorded from Yanbu (subtropical site) largely different from those at Farasan Island mangrove (tropical site) with only six common fungi recorded. Amarenographium solium was the most common fungus at Yanbu mangrove, while Lineolata rhizophorae dominated the fungal community at Farasan Island mangrove. Swampomyces armeniacus was commonly recorded at the two mangrove sites. Torpedospora radiata was the most common species recorded from driftwood samples collected at Yanbu beach. A new collection of Diatrypasimilis australiensis enabled us to make a detailed morphological study of the fungus. Mycelia with chlamydospores were isolated from the samples and subsequently sequenced, and they grouped in the Corollospora clade with various degrees of confidence, but did not sporulate on natural material or in culture.

De novo genome assembly of the soil-borne fungus and tomato pathogen Pyrenochaeta lycopersici.

BackgroundPyrenochaeta lycopersici is a soil-dwelling ascomycete pathogen that causes corky root rot disease in tomato (Solanum lycopersicum) and other Solanaceous crops, reducing fruit yields by up to 75%. Fungal pathogens that infect roots receive less attention than those infecting the aerial parts of crops despite their significant impact on plant growth and fruit production.ResultsWe assembled a 54.9Mb P. lycopersici draft genome sequence based on Illumina short reads, and annotated approximately 17,000 genes. The P. lycopersici genome is closely related to hemibiotrophs and necrotrophs, in agreement with the phenotypic characteristics of the fungus and its lifestyle. Several gene families related to host–pathogen interactions are strongly represented, including those responsible for nutrient absorption, the detoxification of fungicides and plant cell wall degradation, the latter confirming that much of the genome is devoted to the pathogenic activity of the fungus. We did not find a MAT gene, which is consistent with the classification of P. lycopersici as an imperfect fungus, but we observed a significant expansion of the gene families associated with heterokaryon incompatibility (HI).ConclusionsThe P. lycopersici draft genome sequence provided insight into the molecular and genetic basis of the fungal lifestyle, characterizing previously unknown pathogenic behaviors and defining strategies that allow this asexual fungus to increase genetic diversity and to acquire new pathogenic traits.

Genome and transcriptome analysis of the fungal pathogen Fusarium oxysporum f. sp. cubense causing banana vascular wilt disease.

BackgroundThe asexual fungus Fusarium oxysporum f. sp. cubense (Foc) causing vascular wilt disease is one of the most devastating pathogens of banana (Musa spp.). To understand the molecular underpinning of pathogenicity in Foc, the genomes and transcriptomes of two Foc isolates were sequenced.Methodology/Principal FindingsGenome analysis revealed that the genome structures of race 1 and race 4 isolates were highly syntenic with those of F. oxysporum f. sp. lycopersici strain Fol4287. A large number of putative virulence associated genes were identified in both Foc genomes, including genes putatively involved in root attachment, cell degradation, detoxification of toxin, transport, secondary metabolites biosynthesis and signal transductions. Importantly, relative to the Foc race 1 isolate (Foc1), the Foc race 4 isolate (Foc4) has evolved with some expanded gene families of transporters and transcription factors for transport of toxins and nutrients that may facilitate its ability to adapt to host environments and contribute to pathogenicity to banana. Transcriptome analysis disclosed a significant difference in transcriptional responses between Foc1 and Foc4 at 48 h post inoculation to the banana ‘Brazil’ in comparison with the vegetative growth stage. Of particular note, more virulence-associated genes were up regulated in Foc4 than in Foc1. Several signaling pathways like the mitogen-activated protein kinase Fmk1 mediated invasion growth pathway, the FGA1-mediated G protein signaling pathway and a pathogenicity associated two-component system were activated in Foc4 rather than in Foc1. Together, these differences in gene content and transcription response between Foc1 and Foc4 might account for variation in their virulence during infection of the banana variety ‘Brazil’.Conclusions/SignificanceFoc genome sequences will facilitate us to identify pathogenicity mechanism involved in the banana vascular wilt disease development. These will thus advance us develop effective methods for managing the banana vascular wilt disease, including improvement of disease resistance in banana.

Unseen sex in ancient virgin fungi

Unseen sex in ancient virgin fungi

Manglicolous fungi from Chorao mangroves, Goa, West coast of India: Diversity and frequency of occurrence

In this paper marine fungi colonizing the decomposing substrata belonging to Rhizophora mucronata and Avicennia marina, at Chorao mangroves, Mandovi river, Goa, West coast of India, are reported. Totally, 45 fungal species from Mandovi mangroves were recorded from 166 wood and prop root samples: 31 species belonging to Ascomycota, 2 to Basidiomycota and 12 to the asexual fungi. Aigialus grandis (15.3%) was found to be the most frequently occurring fungus followed by Trichocladium achrasporum (8.6%), Morosphaeria ramunculicola (6.7%), Halorosellinia oceanica (6.3%), Rimora mangrovei (5.1%), Dactylospora haliotrepha (5.1%) and Rhizophila marina (5.1%). While 16 species were recorded infrequently (less than 5%), 23 species were 'rare' in their occurrence (less than 1%).

Mating type genes and cryptic sexuality as tools for genetically manipulating industrial molds

A large number of molds serve as producer strains for the industrial production of pharmaceuticals, foods, or organic chemicals. To optimize strains for production processes, conventional strain development programs use random mutagenesis and, more recently, recombinant technologies to generate microbial strains with novel and advantageous properties. The recent detection of mating type genes in fungal production strains and the discovery of cryptic sexuality in presumably asexual fungi open up novel strategies for generating progeny with new, as yet unobserved properties. Mating type genes, which can be considered as "sex genes," not only direct sexual development but also regulate a broad range of fungal secondary metabolites. In addition, they control hyphal morphology, which has a direct impact on production processes that are often conducted in huge fermenter tanks. Here, we survey the occurrence and function of mating type genes that have been discovered in a wide range of industrial fungal producer strains. The possibility to obtain progeny from industrial producers by sexual mating provides an exciting alternative to conventional strain improvement programs aiming to generate optimized recombinant production strains.

Large and variable genome size unrelated to serpentine adaptation but supportive of cryptic sexuality in Cenococcum geophilum

Estimations of genome size and its variation can provide valuable information regarding the genetic diversity of organisms and their adaptation potential to heterogeneous environments. We used flow cytometry to characterize the variation in genome size among 40 isolates of Cenococcum geophilum, an ectomycorrhizal fungus with a wide ecological and geographical distribution, obtained from two serpentine and two non-serpentine sites in Portugal. Besides determining the genome size and its intraspecies variation, we wanted to assess whether a relationship exists between genome size and the edaphic background of the C. geophilum isolates. Our results reveal C. geophilum to have one of the largest genome sizes so far measured in the Ascomycota, with a mean haploid genome size estimate of 0.208pg (203Mbp). However, no relationship was found between genome size and the edaphic background of the sampled isolates, indicating genetic and demographic processes to be more important for shaping the genome size variation in this species than environmental selection. The detection of variation in ploidy level among our isolates, including a single individual with both presumed haploid and diploid nuclei, provides supportive evidence for a possible cryptic sexual or parasexual cycle in C. geophilum (although other mechanisms may have caused this variation). The existence of such a cycle would have wide significance, explaining the high levels of genetic diversity and likelihood of recombination previously reported in this species, and adds to the increasing number of studies suggesting sexual cycles in previously assumed asexual fungi.

Sexual reproduction and mating-type-mediated strain development in the penicillin-producing fungus Penicillium chrysogenum.

Penicillium chrysogenum is a filamentous fungus of major medical and historical importance, being the original and present-day industrial source of the antibiotic penicillin. The species has been considered asexual for more than 100 y, and despite concerted efforts, it has not been possible to induce sexual reproduction, which has prevented sexual crosses being used for strain improvement. However, using knowledge of mating-type (MAT) gene organization, we now describe conditions under which a sexual cycle can be induced leading to production of meiotic ascospores. Evidence of recombination was obtained using both molecular and phenotypic markers. The identified heterothallic sexual cycle was used for strain development purposes, generating offspring with novel combinations of traits relevant to penicillin production. Furthermore, the MAT1-1-1 mating-type gene, known primarily for a role in governing sexual identity, was also found to control transcription of a wide range of genes with biotechnological relevance including those regulating penicillin production, hyphal morphology, and conidial formation. These discoveries of a sexual cycle and MAT gene function are likely to be of broad relevance for manipulation of other asexual fungi of economic importance.

Occurrence of microfungi as litter colonizers and endophytes in varied plant species from the Western Ghats forests, Goa, India

In this study 30 widely distributed plant species from the Western Ghat forest in Goa were randomly selected and were studied with regard to their fungal association as endophytes and litter colonizers. This effort resulted in the recovery of more than 6500 isolates of microfungi which were assignable to 675 species of fungi belonging to 275 genera which included properly recognized Mucorales(1), Ascomycetes (18), Hyphomycetous asexual fungi (289), Coelomycetous asexual fungi (22) and undetermined taxa (77), besides a sizable number of non-sporulating forms (268). Species of endophytes (53) and litter colonizers (77) were selected for enzyme studies. Ten taxa occurred both as endophytes and litter colonizers. Endophytes and litter colonizers showed different enzyme profiles indicating that habit and habitat dictated enzyme activity. Several recovered fungi were new to science and some have already been described as new species and are elaborated here in this paper. Some taxa showed substrate specificity others were diverse in their distribution. Not a single taxon was found to occur on all 26 plant species and only three taxa showed more than 50% association with all the 26 plant species. This shows evidence towards substrate preference in fungi in relation to their host plants.

A checklist of marine fungi of Taiwan with a description of Kitesporella keelungensis gen. et sp. nov

Abstract Taiwan is an island of 35,980 km2 with a long coastline and thus is an ideal place for the study of marine fungi. A literature search and continuous surveys of lignicolous marine fungi over the past few years have documented 107 taxa: 2 Basidiomycota, 94 Ascomycota and 11 asexual fungi (10 hyphomycetes, 1 coelomycete). Most of these taxa were discovered on wood with a few arenicolous species. One new marine lignicolous fungus belonging to the Halosphaeriaceae was encountered during our study. Kitesporella keelungensis gen. et sp. nov. is characterized by hyaline, coriaceous ascomata with short necks, absence of periphyses and catenophyses, persistent/semi-persistent, clavate asci and rhomboid-shaped, thin-walled ascospores without appendages. It resembles Remispora pilleata in the rhomboid shape of the ascospores, but in R. pilleata, periphyses are present, asci are deliquescent and ascospores thick-walled with pleomorphic polar appendages at both ends. We compare the morphology of Kitesporella with those of other taxa in the Halosphaeriaceae without appendaged ascospores.

A novel selectable marker based on Aspergillus niger arginase expression

Selectable markers are valuable tools in transforming asexual fungi like Aspergillus niger. An arginase (agaA) expression vector and a suitable arginase-disrupted host would define a novel nutritional marker/selection for transformation. The development of such a marker was successfully achieved in two steps. The single genomic copy of A. niger arginase gene was disrupted by homologous integration of the bar marker. The agaA disruptant was subsequently complemented by transforming it with agaA expression vectors. Both citA and trpC promoters were able to drive the expression of arginase cDNA. Such agaA+ transformants displayed arginase expression pattern distinct from that of the parent strain. The results are also consistent with a single catabolic route for arginine in this fungus. A simple yet novel arginine-based selection for filamentous fungal transformation is thus described.

Mutualism effectiveness and vertical transmission of symbiotic fungal endophytes in response to host genetic background

Certain species of the Pooideae subfamily develop stress tolerance and herbivory resistance through symbiosis with vertically transmitted, asexual fungi. This symbiosis is specific, and genetic factors modulate the compatibility between partners. Although gene flow is clearly a fitness trait in allogamous grasses, because it injects hybrid vigor and raw material for evolution, it could reduce compatibility and thus mutualism effectiveness. To explore the importance of host genetic background in modulating the performance of symbiosis, Lolium multiflorum plants, infected and noninfected with Neotyphodium occultans, were crossed with genetically distant plants of isolines (susceptible and resistant to diclofop-methyl herbicide) bred from two cultivars and exposed to stress. The endophyte improved seedling survival in genotypes susceptible to herbicide, while it had a negative effect on one of the genetically resistant crosses. Mutualism provided resistance to herbivory independently of the host genotype, but this effect vanished under stress. While no endophyte effect was observed on host reproductive success, it was increased by interpopulation plant crosses. Neither gene flow nor herbicide had an important impact on endophyte transmission. Host fitness improvements attributable to gene flow do not appear to result in direct conflict with mutualism while this seems to be an important mechanism for the ecological and contemporary evolution of the symbiotum.

Sex, drugs and recombination: the wild life of Aspergillus

Throughout the eukaryotes, sexual reproduction is an almost universal phenomenon. However, within the Kingdom Fungi, this relationship is not so clear-cut. Fungi exhibit a spectrum of reproductive modes and life-cycles; amongst the better known species, sexual reproduction is often facultative, can be rare, and in over half of the known Ascomycota (the moulds) is unknown (Taylor etal. 1999). However, over the last decade, it has become apparent that many of these asexual mitosporic taxa undergo cryptic recombination via unobserved mechanisms and that wholly asexual fungi are, in fact, a rarity (Taylor etal. 1999, 2001; Heitman 2010). This revolution in our understanding of fungal sexuality has come about in two ways: Firstly, sexual reproduction leaves an imprint on fungal genomes by maintaining genes required for mating and by generating patterns of mutation and recombination restricted to meiotic processes. Secondly, scientists have become better at catching fungi in flagrante delicto. The genus Aspergillus is one such fungus where a combination of population genetics, genomics and taxonomy has been able to intuit the existence of sex, then to catch the fungus in the act and formally describe their sexual stages. So, why are sexy moulds exciting? One species in particular, Aspergillus flavus, is notorious for its ability to produce a diverse array of secondary metabolites, of which the polyketide aflatoxins (AF) are carcinogenic and others (such as cyclopiazonic acid) are toxigenic. Because of the predilection of A.flavus to grow on crops, such as peanuts, corn and cotton, biocontrol is widely used to mitigate infection by pre-applying nonaflatoxigenic (AF-) strains to competitively exclude the wild-type AF+ strains. However, the eventual fate in nature of these biocontrol strains is not known. In this issue of Molecular Ecology, Olarte etal. (2012) make an important contribution by using laboratory crosses of A.flavus to show that not only is AF highly heritable, but AF- strains can become AF+ via crossing over during meiosis. This observation has raised the spectre of cross-breeding and non-mendelian inheritance of AF between native and biocontrol strains of the fungus, leading to an increase in the natural diversity of the fungus with perhaps unanticipated consequences.

Presence and Functionality of Mating Type Genes in the Supposedly Asexual Filamentous Fungus Aspergillus oryzae

The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the reference strain RIB40. We now report the existence of a complementary MAT1-2 gene and the sequencing of an idiomorphic region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of the MAT1-1 and MAT1-2 genotypes among 180 strains assayed, including industrial tane-koji isolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in which MAT genes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both the MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating type-dependent manner, the first such report for a supposedly asexual fungus. MAT1-1 expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed.

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.

Genetic Diversity, Recombination, and Divergence in Animal Associated Penicillium dipodomyis

Penicillium dipodomyis is thought to be an exclusively asexual fungus associated with Kangaroo Rats, Dipodomys species, and is unique among Penicillium species in growing at 37°C but producing no known toxins. Lack of recombination within P. dipodomyis would result in limited adaptive flexibility but possibly enhance local adaptation and host selection via maintenance of favourable genotypes. Here, analysis of DNA sequence data from five protein-coding genes shows that recombination occurs within P. dipodomyis on a small spatial scale. Furthermore, detection of mating-type alleles supports outcrossing and a sexual cycle in P. dipodomyis. P. dipodomyis was a weaker competitor in in vitro assays with other Penicillium species found in association with Kanagaroo rats. Bayesian species level analysis suggests that the P. dipodomyis lineage diverged from closely related species also found in cheek pouches of Kangaroo Rats and their stored seeds about 11 million years ago, a similar divergence time as Dipodomys from its sister rodent taxa.