Publications by authors named "Brenda D Wingfield"

141 Publications

Unidirectional mating-type switching confers self-fertility to Thielaviopsis cerberus, the only homothallic species in the genus.

Fungal Biol 2021 Jun 7;125(6):427-434. Epub 2021 Jan 7.

Department of Biochemistry, Genetics and Microbiology; Forestry and Agricultural Biotechnology Institute, University of Pretoria, Private Bag X20, Hatfield, Pretoria, 0028, South Africa. Electronic address:

Sexual reproduction is ubiquitous in nature, and nowhere is this more so than in the fungi. Heterothallic behaviour is observed when there is a strict requirement of contact between two individuals of opposite mating type for sexual reproduction to occur. In contrast, a homothallic species can complete the entire sexual cycle in isolation, although several genetic mechanisms underpin this self-fertility. These can be inferred by characterising the structure and gene-content of the mating-type locus, which contains genes that are involved in the regulation of sexual reproduction. In this study, the genetic basis of homothallism in Thielaviopsis cerberus was investigated, the only known self-fertile species within this genus. Using genome sequencing and conventional molecular techniques, two versions of the mating-type locus were identified in this species. This is typical of species that have a unidirectional mating-type switching reproductive strategy. The first version was a self-fertile locus that contained four known mating-type genes, while the second was a self-sterile version with a single mating-type gene. The conversion from a self-fertile to a self-sterile locus is likely mediated by a homologous recombination event at two direct repeats present in the self-fertile locus, resulting in the deletion of three mating-type genes and one of the repeats. Both locus versions were present in isolates that were self-fertile, while self-sterility was caused by the presence of only a switched locus. This study provides a clear example of the architectural fluidity in the mating-type loci that is common among even closely related fungal species.
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http://dx.doi.org/10.1016/j.funbio.2020.12.007DOI Listing
June 2021

Characterization of the Ergosterol Biosynthesis Pathway in Ceratocystidaceae.

J Fungi (Basel) 2021 Mar 22;7(3). Epub 2021 Mar 22.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa.

Terpenes represent the biggest group of natural compounds on earth. This large class of organic hydrocarbons is distributed among all cellular organisms, including fungi. The different classes of terpenes produced by fungi are mono, sesqui, di- and triterpenes, although triterpene ergosterol is the main sterol identified in cell membranes of these organisms. The availability of genomic data from members in the Ceratocystidaceae enabled the detection and characterization of the genes encoding the enzymes in the mevalonate and ergosterol biosynthetic pathways. Using a bioinformatics approach, fungal orthologs of sterol biosynthesis genes in nine different species of the Ceratocystidaceae were identified. Ergosterol and some of the intermediates in the pathway were also detected in seven species (, , , , , and ), using gas chromatography-mass spectrometry analysis. The average ergosterol content differed among different genera of Ceratocystidaceae. We also identified all possible terpene related genes and possible biosynthetic clusters in the genomes used in this study. We found a highly conserved terpene biosynthesis gene cluster containing some genes encoding ergosterol biosynthesis enzymes in the analysed genomes. An additional possible terpene gene cluster was also identified in all of the Ceratocystidaceae. We also evaluated the sensitivity of the Ceratocystidaceae to a triazole fungicide that inhibits ergosterol synthesis. The results showed that different members of this family behave differently when exposed to different concentrations of triazole tebuconazole.
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http://dx.doi.org/10.3390/jof7030237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004197PMC
March 2021

Armillaria root rot fungi host single-stranded RNA viruses.

Sci Rep 2021 Apr 1;11(1):7336. Epub 2021 Apr 1.

Natural Resources Institute Finland (Luke), Helsinki, Finland.

Species of Armillaria are distributed globally and include some of the most important pathogens of forest and ornamental trees. Some of them form large long-living clones that are considered as one of the largest organisms on earth and are capable of long-range spore-mediated transfer as well as vegetative spread by drought-resistant hyphal cords called rhizomorphs. However, the virus community infecting these species has remained unknown. In this study we used dsRNA screening and high-throughput sequencing to search for possible virus infections in a collection of Armillaria isolates representing three different species: Armillaria mellea from South Africa, A. borealis from Finland and Russia (Siberia) and A. cepistipes from Finland. Our analysis revealed the presence of both negative-sense RNA viruses and positive-sense RNA viruses, while no dsRNA viruses were detected. The viruses included putative new members of virus families Mymonaviridae, Botourmiaviridae and Virgaviridae and members of a recently discovered virus group tentatively named "ambiviruses" with ambisense bicistronic genomic organization. We demonstrated that Armillaria isolates can be cured of viruses by thermal treatment, which enables the examination of virus effects on host growth and phenotype using isogenic virus-infected and virus-free strains.
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http://dx.doi.org/10.1038/s41598-021-86343-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016926PMC
April 2021

IMA genome - F14 : Draft genome sequences of Penicillium roqueforti, Fusarium sororula, Chrysoporthe puriensis, and Chalaropsis populi.

IMA Fungus 2021 Mar 5;12(1). Epub 2021 Mar 5.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private bag X20, Pretoria, 0028, South Africa.

Draft genomes of Penicillium roqueforti, Fusarium sororula, Chalaropsis populi, and Chrysoporthe puriensis are presented. Penicillium roqueforti is a model fungus for genetics, physiological and metabolic studies, as well as for biotechnological applications. Fusarium sororula and Chrysoporthe puriensis are important tree pathogens, and Chalaropsis populi is a soil-borne root-pathogen. The genome sequences presented here thus contribute towards a better understanding of both the pathogenicity and biotechnological potential of these species.
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http://dx.doi.org/10.1186/s43008-021-00055-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934431PMC
March 2021

Genome-Wide Analyses of Repeat-Induced Point Mutations in the Ascomycota.

Front Microbiol 2020 1;11:622368. Epub 2021 Feb 1.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.

The Repeat-Induced Point (RIP) mutation pathway is a fungus-specific genome defense mechanism that mitigates the deleterious consequences of repeated genomic regions and transposable elements (TEs). RIP mutates targeted sequences by introducing cytosine to thymine transitions. We investigated the genome-wide occurrence and extent of RIP with a sliding-window approach. Using genome-wide RIP data and two sets of control groups, the association between RIP, TEs, and GC content were contrasted in organisms capable and incapable of RIP. Based on these data, we then set out to determine the extent and occurrence of RIP in 58 representatives of the Ascomycota. The findings were summarized by placing each of the fungi investigated in one of six categories based on the extent of genome-wide RIP. RIP analyses, using a sliding-window approach with stringent RIP parameters, implemented simultaneously within the same genetic context, on high quality genome assemblies, yielded superior results in determining the genome-wide RIP among the Ascomycota. Most Ascomycota had RIP and these mutations were particularly widespread among classes of the Pezizomycotina, including the early diverging Orbiliomycetes and the Pezizomycetes. The most extreme cases of RIP were limited to representatives of the Dothideomycetes and Sordariomycetes. By contrast, the genomes of the Taphrinomycotina and Saccharomycotina contained no detectable evidence of RIP. Also, recent losses in RIP combined with controlled TE proliferation in the Pezizomycotina subphyla may promote substantial genome enlargement as well as the formation of sub-genomic compartments. These findings have broadened our understanding of the taxonomic range and extent of RIP in Ascomycota and how this pathway affects the genomes of fungi harboring it.
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http://dx.doi.org/10.3389/fmicb.2020.622368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7882544PMC
February 2021

IMA Genome - F13: Draft genome sequences of , and .

IMA Fungus 2020 24;11:19. Epub 2020 Sep 24.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag x20, Hatfield, Pretoria, 0028 South Africa.

Draft genomes of the fungal species , and are presented. is an important lichen forming fungus and is an ambrosia beetle symbiont. and are agriculturally relevant plant pathogens that cause leaf-spots in brassicaceous vegetables and cucurbits respectively. causes severe leaf blight and defoliation of trees. These genomes provide a valuable resource for understanding the molecular processes in these economically important fungi.
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http://dx.doi.org/10.1186/s43008-020-00039-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513301PMC
September 2020

Genome comparisons suggest an association between Ceratocystis host adaptations and effector clusters in unique transposable element families.

Fungal Genet Biol 2020 10 8;143:103433. Epub 2020 Jul 8.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa. Electronic address:

Ceratocystis fimbriata is a host specific fungal pathogen of sweet potato (Ipomoea batatas). The closely related species, C. manginecans, is an important pathogen of trees (e.g. Acacia mangium and Mangifera indica) but has never been isolated from tuber crops. The genetic factors that determine the host range and host specificity of these species have not been determined. The aim of this study was to compare the genomes of C. fimbriata and C. manginecans in order to identify species-specific genetic differences that could be associated with host specificity. This included whole-genome alignments as well as comparisons of gene content and transposable elements (TEs). The genomes of the two species were found to be very similar, sharing similar catalogues of CAZymes, peptidases and lipases. However, the genomes of the two species also varied, harbouring species-specific genes (e.g. small secreted effectors, nutrient processing proteins and stress response proteins). A portion of the TEs identified (17%) had a unique distribution in each species. Transposable elements appeared to have played a prominent role in the divergence of the two species because they were strongly associated with chromosomal translocations and inversions as well as with unique genomic regions containing species-specific genes. Two large effector clusters, with unique TEs in each species, were identified. These effectors displayed non-synonymous mutations and deletions, conserved within a species, and could serve as mutational hot-spots for the development of host specificity in the two species.
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http://dx.doi.org/10.1016/j.fgb.2020.103433DOI Listing
October 2020

Draft genome sequences of five species from plantations in China, , and .

IMA Fungus 2019 27;10:22. Epub 2019 Dec 27.

Department of Biochemistry, Genetics and Microbiology (BGM), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0028 South Africa.

Draft genome sequences of five species [including , , , and ], , and are presented. Species of are the causal agents of Eucalyptus leaf blight disease, threatening the growth and sustainability of plantations in China. is the causal agent of stem canker in native and exotic in South Africa. was first discovered in the infructescences of and in South Africa. is fruticose lichen belongs to the alectorioid clade of the family Parmeliaceae. The availability of these genome sequences will facilitate future studies on the systematics, population genetics, and genomics of these fungi.
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http://dx.doi.org/10.1186/s43008-019-0023-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325655PMC
December 2019

Fungal clones win the battle, but recombination wins the war.

IMA Fungus 2019 29;10:18. Epub 2019 Oct 29.

Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag X20, Pretoria, Gauteng South Africa.

Clonal reproduction is common in fungi and fungal-like organisms during epidemics and invasion events. The success of clonal fungi shaped systems for their classification and some pathogens are tacitly treated as asexual. We argue that genetic recombination driven by sexual reproduction must be a starting hypothesis when dealing with fungi for two reasons: (1) Clones eventually crash because they lack adaptability; and (2) fungi find a way to exchange genetic material through recombination, whether sexual, parasexual, or hybridisation. Successful clones may prevail over space and time, but they are the product of recombination and the next successful clone will inevitably appear. Fungal pathogen populations are dynamic rather than static, and they need genetic recombination to adapt to a changing environment.
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http://dx.doi.org/10.1186/s43008-019-0020-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325676PMC
October 2019

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis.

J Vis Exp 2020 06 9(160). Epub 2020 Jun 9.

Department of Biochemistry, Genetics & Microbiology, University of Pretoria; Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria.

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.
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http://dx.doi.org/10.3791/61367DOI Listing
June 2020

Phylogenomic incongruence in Ceratocystis: a clue to speciation?

BMC Genomics 2020 May 14;21(1):362. Epub 2020 May 14.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa.

Background: The taxonomic history of Ceratocystis, a genus in the Ceratocystidaceae, has been beset with questions and debate. This is due to many of the commonly used species recognition concepts (e.g., morphological and biological species concepts) providing different bases for interpretation of taxonomic boundaries. Species delineation in Ceratocystis primarily relied on genealogical concordance phylogenetic species recognition (GCPSR) using multiple standard molecular markers.

Results: Questions have arisen regarding the utility of these markers e.g., ITS, BT and TEF1-α due to evidence of intragenomic variation in the ITS, as well as genealogical incongruence, especially for isolates residing in a group referred to as the Latin-American clade (LAC) of the species. This study applied a phylogenomics approach to investigate the extent of phylogenetic incongruence in Ceratocystis. Phylogenomic analyses of a total of 1121 shared BUSCO genes revealed widespread incongruence within Ceratocystis, particularly within the LAC, which was typified by three equally represented topologies. Comparative analyses of the individual gene trees revealed evolutionary patterns indicative of hybridization. The maximum likelihood phylogenetic tree generated from the concatenated dataset comprised of 1069 shared BUSCO genes provided improved phylogenetic resolution suggesting the need for multiple gene markers in the phylogeny of Ceratocystis.

Conclusion: The incongruence observed among single gene phylogenies in this study call into question the utility of single or a few molecular markers for species delineation. Although this study provides evidence of interspecific hybridization, the role of hybridization as the source of discordance will require further research because the results could also be explained by high levels of shared ancestral polymorphism in this recently diverged lineage. This study also highlights the utility of BUSCO genes as a set of multiple orthologous genes for phylogenomic studies.
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http://dx.doi.org/10.1186/s12864-020-6772-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7222570PMC
May 2020

IMA Genome-F 11: Draft genome sequences of and and genome annotation for .

IMA Fungus 2019 13;10:13. Epub 2019 Sep 13.

1Department of Biochemistry, Genetics and Microbiology (BGM), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag X20, Hatfield, 0028 South Africa.

Draft genomes of the fungal species and are presented. In addition an annotation of the genome of is presented. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity and potential management strategies of these economically important fungi.
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http://dx.doi.org/10.1186/s43008-019-0013-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184890PMC
September 2019

Quantification of Outcrossing Events in Haploid Fungi Using Microsatellite Markers.

J Fungi (Basel) 2020 Apr 14;6(2). Epub 2020 Apr 14.

Department of Biochemistry, Genetics, and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0028, South Africa.

Species in genera of the fungal family Ceratocystidaceae are known to have different mating strategies, including heterothallism and homothallism. Of these, species of , typified by the pathogen all undergo unidirectional mating-type switching. This implies that the pathogens possess the ability to self, but also to undergo sexual outcrossing between isolates of different mating types. In this study, we extended the recently developed microsatellite-based technique to determine the extent to which outcrossing occurs in ascospore masses of haploid fungi to two field collections of . In this way, the role of reproductive strategies in shaping population structure and diversity could be better understood. Results showed that a high frequency of outcrossing occurs in isolates of the pathogen from both non-native and native areas. This explains the high level of genetic diversity previously observed in this population despite the fact that this pathogen has the ability to self.
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http://dx.doi.org/10.3390/jof6020048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7345254PMC
April 2020

Mating strategy and mating type distribution in six global populations of the Eucalyptus foliar pathogen Teratosphaeria destructans.

Fungal Genet Biol 2020 04 31;137:103350. Epub 2020 Jan 31.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag X20, Hatfield 0028, South Africa; Department of Conservation Ecology and Entomology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa. Electronic address:

Teratosphaeria destructans is an aggressive fungal pathogen causing leaf and shoot blight on young Eucalyptus trees in plantations. The disease occurs across tropical and subtropical regions of South East Asia and has recently been found in South Africa. Asexual structures of the pathogen are produced on infected tissues, but sexual structures have never been observed. The aim of this study was to investigate the reproductive biology of T. destructans by characterising its mating type (MAT1) locus and investigating its potential for sexual recombination. We found that T. destructans has a heterothallic mating system, with either the MAT1-1-1 and MAT1-1-10 genes (MAT1-1 idiomorph) or the MAT1-2-1 and MAT1-2-12 genes (MAT1-2 idiomorph) present in a single individual. With a multiplex PCR assay, it was possible to distinguish the two MAT idiomorphs in several Teratosphaeria species and this approach was applied to six global populations of T. destructans. Although both mating types occurred in the South East Asian populations, a single mating type dominated each population. Isolates from the recent disease outbreak in South Africa comprised only a single mating type. Attempts to induce a sexual cycle in vitro using strains of opposite mating type were not successful. The uneven distribution of mating types in populations of T. destructans and the presence of only an asexual state on infected tissues suggests the absence of or at least a minor role for sexual reproduction where the pathogen occurs on non-native Eucalyptus in plantations.
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http://dx.doi.org/10.1016/j.fgb.2020.103350DOI Listing
April 2020

The novel Huntiella omanensis mating gene, MAT1-2-7, is essential for ascomatal maturation.

Fungal Genet Biol 2020 04 17;137:103335. Epub 2020 Jan 17.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), Faculty of Natural and Agricultural Sciences, University of Pretoria, Hatfield Campus, Pretoria 0083, South Africa. Electronic address:

Sexual reproduction is a highly conserved feature of the eukaryotes, yet sexual compatibility is determined by a wide variety of mechanisms. In ascomycete fungi, sexual development is controlled by genes at the mating type (MAT) locus that confer either MAT1-1 or MAT1-2 mating identity. Although the locus harbours, at minimum, a single gene, the individual MAT loci of certain species, including Huntiella omanensis, encode for two or more genes. The MAT1-2 idiomorph of H. omanensis is made up of MAT1-2-1, a primary MAT gene that is highly conserved in the Pezizomycotina and possesses a well-characterized DNA binding motif, the HMG-box domain. The idiomorph also harbours a novel secondary MAT gene, named MAT1-2-7, with no recognizable functional domains. In this study, we developed a transformation and CRISPR-Cas9-based genome editing protocol to characterize the MAT1-2-7 gene with respect to its function in mating. We have shown that MAT1-2-7 is essential for sexual reproduction and that isolates carrying the truncated MAT1-2-7 gene are incapable of ascomatal maturation and further sexual development. MAT1-2-7 was also shown to influence the vegetative radial growth rate of H. omanensis, illustrating the pleiotropic effects often associated with MAT genes.
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http://dx.doi.org/10.1016/j.fgb.2020.103335DOI Listing
April 2020

Repeat-Induced Point Mutations Drive Divergence between and Its Close Relatives.

Pathogens 2019 Dec 14;8(4). Epub 2019 Dec 14.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag X20, Pretoria 0028, South Africa.

The Repeat-Induced Point (RIP) mutation pathway is a fungal-specific genome defense mechanism that counteracts the deleterious effects of transposable elements. This pathway permanently mutates its target sequences by introducing cytosine to thymine transitions. We investigated the genome-wide occurrence of RIP in the pitch canker pathogen, , and its close relatives in the species complex (FFSC). Our results showed that the examined fungi all exhibited hallmarks of RIP, but that they differed in terms of the extent to which their genomes were affected by this pathway. RIP mutations constituted a large proportion of all the FFSC genomes, including both core and dispensable chromosomes, although the latter were generally more extensively affected by RIP. Large RIP-affected genomic regions were also much more gene sparse than the rest of the genome. Our data further showed that RIP-directed sequence diversification increased the variability between homologous regions of related species, and that RIP-affected regions can interfere with homologous recombination during meiosis, thereby contributing to post-mating segregation distortion. Taken together, these findings suggest that RIP can drive the independent divergence of chromosomes, alter chromosome architecture, and contribute to the divergence among and other members of this economically important group of fungi.
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http://dx.doi.org/10.3390/pathogens8040298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963459PMC
December 2019

The RIPper, a web-based tool for genome-wide quantification of Repeat-Induced Point (RIP) mutations.

PeerJ 2019 26;7:e7447. Epub 2019 Aug 26.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, Gauteng, South Africa.

Background: The RIPper (http://theripper.hawk.rocks) is a set of web-based tools designed for analyses of Repeat-Induced Point (RIP) mutations in the genome sequences of Ascomycota. The RIP pathway is a fungal genome defense mechanism that is aimed at identifying repeated and duplicated motifs, into which it then introduces cytosine to thymine transition mutations. RIP thus serves to deactivate and counteract the deleterious consequences of selfish or mobile DNA elements in fungal genomes. The occurrence, genetic context and frequency of RIP mutations are widely used to assess the activity of this pathway in genomic regions of interest. Here, we present a bioinformatics tool that is specifically fashioned to automate the investigation of changes in RIP product and substrate nucleotide frequencies in fungal genomes.

Results: We demonstrated the ability of The RIPper to detect the occurrence and extent of RIP mutations in known RIP affected sequences. Specifically, a sliding window approach was used to perform genome-wide RIP analysis on the genome assembly of . Additionally, fine-scale analysis with The RIPper showed that gene regions and transposable element sequences, previously determined to be affected by RIP, were indeed characterized by high frequencies of RIP mutations. Data generated using this software further showed that large proportions of the genome constitutes RIP mutations with extensively affected regions displaying reduced GC content. The RIPper was further useful for investigating and visualizing changes in RIP mutations across the length of sequences of interest, allowing for fine-scale analyses.

Conclusion: This software identified RIP targeted genomic regions and provided RIP statistics for an entire genome assembly, including the genomic proportion affected by RIP. Here, we present The RIPper as an efficient tool for genome-wide RIP analyses.
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http://dx.doi.org/10.7717/peerj.7447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714961PMC
August 2019

QTL mapping of mycelial growth and aggressiveness to distinct hosts in Ceratocystis pathogens.

Fungal Genet Biol 2019 10 15;131:103242. Epub 2019 Jun 15.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa. Electronic address:

Some species of Ceratocystis display strong host specificity, such as C. fimbriata sensu stricto that is restricted to sweet potato (Ipomoea batatas) as host. In contrast, the closely related C. manginecans, infects Acacia mangium and Mangifera indica but is not pathogenic to I. batatas. Despite the economic importance of these fungi, knowledge regarding the genetic factors that influence their pathogenicity and host specificity is limited. A recent inheritance study, based on an interspecific cross between C. fimbriata and C. manginecans and the resultant 70 F progeny, confirmed that traits such as mycelial growth rate, spore production and aggressiveness on A. mangium and I. batatas are regulated by multiple genes. In the present study, a quantitative trait locus (QTL) analysis was performed to determine the genomic loci associated with these traits. All 70 progeny isolates were genotyped with SNP markers and a linkage map was constructed. The map contained 467 SNPs, distributed across nine linkage groups, with a total length of 1203 cm. Using the progeny genotypes and phenotypes, one QTL was identified on the linkage map for mycelial growth rate, one for aggressiveness to A. mangium and two for aggressiveness to I. batatas (P < 0.05). Two candidate genes, likely associated with mycelial growth rate, were identified in the QTL region. The three QTLs associated with aggressiveness to different hosts contained candidate genes involved in protein processing, detoxification and regions with effector genes and high transposable element density. The results provide a foundation for studies considering the function of genes regulating various quantitative traits in Ceratocystis.
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http://dx.doi.org/10.1016/j.fgb.2019.103242DOI Listing
October 2019

Genomic analysis of the aggressive tree pathogen Ceratocystis albifundus.

Fungal Biol 2019 05 16;123(5):351-363. Epub 2019 Feb 16.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.

The overall goal of this study was to determine whether the genome of an important plant pathogen in Africa, Ceratocystis albifundus, is structured into subgenomic compartments, and if so, to establish how these compartments are distributed across the genome. For this purpose, the publicly available genome of C. albifundus was complemented with the genome sequences for four additional isolates using the Illumina HiSeq platform. In addition, a reference genome for one of the individuals was assembled using both PacBio and Illumina HiSeq technologies. Our results showed a high degree of synteny between the five genomes, although several regions lacked detectable long-range synteny. These regions were associated with the presence of accessory genes, lower genetic similarity, variation in read-map depth, as well as transposable elements and genes associated with host-pathogen interactions (e.g. effectors and CAZymes). Such patterns are regarded as hallmarks of accelerated evolution, particularly of accessory subgenomic compartments in fungal pathogens. Our findings thus showed that the genome of C. albifundus is made-up of core and accessory subgenomic compartments, which is an important step towards characterizing its pangenome. This study also highlights the value of comparative genomics for understanding mechanisms that may underly and influence the biology and evolution of pathogens.
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http://dx.doi.org/10.1016/j.funbio.2019.02.002DOI Listing
May 2019

It's All in the Genes: The Regulatory Pathways of Sexual Reproduction in Filamentous Ascomycetes.

Genes (Basel) 2019 04 30;10(5). Epub 2019 Apr 30.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria 0083, South Africa.

Sexual reproduction in filamentous ascomycete fungi results in the production of highly specialized sexual tissues, which arise from relatively simple, vegetative mycelia. This conversion takes place after the recognition of and response to a variety of exogenous and endogenous cues, and relies on very strictly regulated gene, protein, and metabolite pathways. This makes studying sexual development in fungi an interesting tool in which to study gene-gene, gene-protein, and protein-metabolite interactions. This review provides an overview of some of the most important genes involved in this process; from those involved in the conversion of mycelia into sexually-competent tissue, to those involved in the development of the ascomata, the asci, and ultimately, the ascospores.
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http://dx.doi.org/10.3390/genes10050330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562746PMC
April 2019

Distribution and Evolution of Nonribosomal Peptide Synthetase Gene Clusters in the .

Genes (Basel) 2019 04 30;10(5). Epub 2019 Apr 30.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria 0002, South Africa.

In filamentous fungi, genes in secondary metabolite biosynthetic pathways are generally clustered. In the case of those pathways involved in nonribosomal peptide production, a nonribosomal peptide synthetase (NRPS) gene is commonly found as a main element of the cluster. Large multifunctional enzymes are encoded by members of this gene family that produce a broad spectrum of bioactive compounds. In this research, we applied genome-based identification of nonribosomal peptide biosynthetic gene clusters in the family . For this purpose, we used the whole genome sequences of species from the genera , , and . To identify and characterize the clusters, different bioinformatics and phylogenetic approaches, as well as PCR-based methods were used. In all genomes studied, two highly conserved NRPS genes (one monomodular and one multimodular) were identified and their potential products were predicted to be siderophores. Expression analysis of two species ( and ) confirmed the accuracy of the annotations and proved that the genes in both clusters are expressed. Furthermore, a phylogenetic analysis showed that both NRPS genes of the formed distinct and well supported clades in their respective phylograms, where they grouped with other known NRPSs involved in siderophore production. Overall, these findings improve our understanding of the diversity and evolution of NRPS biosynthetic pathways in the family .
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http://dx.doi.org/10.3390/genes10050328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563098PMC
April 2019

Nine draft genome sequences of ., including , and cf. pseudomolecules for the pitch canker pathogen , draft genome of and .

IMA Fungus 2018 Jul 14;9:401-418. Epub 2018 Dec 14.

Department of Biochemistry, Genetics and Microbiology (BGM), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag x20, Hatfield, Pretoria, 0028, South Africa.

This genome announcement includes draft genomes from including , and cf. The draft genomes of and all three important eucalyptus pathogens, are presented. The insect associate is also described. The pine pathogen genome of has been assembled into pseudomolecules, based on additional sequence data and by harnessing the known synteny within the species complex. This new assembly of the genome provides 12 pseudomolecules that correspond to the haploid chromosome number of . These are comparable to other chromosomal assemblies within the FFSC and will enable more robust genomic comparisons within this species complex.
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http://dx.doi.org/10.5598/imafungus.2018.09.02.10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317589PMC
July 2018

The mating system of the Eucalyptus canker pathogen Chrysoporthe austroafricana and closely related species.

Fungal Genet Biol 2019 02 4;123:41-52. Epub 2018 Dec 4.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa.

Fungi in the genus Chrysoporthe are economically important canker pathogens of commercially grown Eucalyptus species and native Myrtales. Before the current study, homothallism was widely accepted as the mating system of these species, but this hypothesis could not be fully tested. Using whole genome sequences, we characterized the MAT locus of two C. austroafricana isolates and its sibling species, C. cubensis and C. deuterocubensis. A unique MAT1-2 idiomorph containing a truncated MAT1-1-1 gene, and a MAT1-1-2 gene, was identified in one isolate of C. austroafricana and a MAT1-1 idiomorph was found in the other. The presence of a single idiomorph in each isolate suggests that this fungus is heterothallic. Screening for MAT genes in 65 C. austroafricana isolates revealed a bias towards MAT1-2 idiomorphs suggesting a recent introduction in Eucalyptus species. Chrysoporthe cubensis and C. deuterocubensis are apparently homothallic since all the expected MAT genes were identified in their genome sequences. These findings were corroborated by the expression profiles of pheromone genes and their receptors, which conformed to the expected patterns observed in heterothallic and homothallic isolates. Long terminal repeat sequences (LTRs) and specifically retrotransposons were identified in the MAT locus of C. deuterocubensis and C. cubensis, indicating that the evolution of mating systems in Chrysoporthe species could be mediated by these elements.
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http://dx.doi.org/10.1016/j.fgb.2018.12.001DOI Listing
February 2019

Genomic overview of closely related fungi with different Protea host ranges.

Fungal Biol 2018 12 23;122(12):1201-1214. Epub 2018 Oct 23.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa.

Genome comparisons of species with distinctive ecological traits can elucidate genetic divergence that influenced their differentiation. The interaction of a microorganism with its biotic environment is largely regulated by secreted compounds, and these can be predicted from genome sequences. In this study, we considered Knoxdaviesia capensis and Knoxdaviesia proteae, two closely related saprotrophic fungi found exclusively in Protea plants. We investigated their genome structure to compare their potential inter-specific interactions based on gene content. Their genomes displayed macrosynteny and were approximately 10 % repetitive. Both species had fewer secreted proteins than pathogens and other saprotrophs, reflecting their specialized habitat. The bulk of the predicted species-specific and secreted proteins coded for carbohydrate metabolism, with a slightly higher number of unique carbohydrate-degrading proteins in the broad host-range K. capensis. These fungi have few secondary metabolite gene clusters, suggesting minimal competition with other microbes and symbiosis with antibiotic-producing bacteria common in this niche. Secreted proteins associated with detoxification and iron sequestration likely enable these Knoxdaviesia species to tolerate antifungal compounds and compete for resources, facilitating their unusual dominance. This study confirms the genetic cohesion between Protea-associated Knoxdaviesia species and reveals aspects of their ecology that have likely evolved in response to their specialist niche.
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http://dx.doi.org/10.1016/j.funbio.2018.10.001DOI Listing
December 2018

Ceratocystidaceae exhibit high levels of recombination at the mating-type (MAT) locus.

Fungal Biol 2018 12 22;122(12):1184-1191. Epub 2018 Sep 22.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, 0002, South Africa. Electronic address:

Mating is central to many fungal life cycles and is controlled by genes at the mating-type (MAT) locus. These genes determine whether the fungus will be self-sterile (heterothallic) or self-fertile (homothallic). Species in the ascomycete family Ceratocystidaceae have different mating strategies, making them interesting to consider with regards to their MAT loci. The aim of this study was to compare the composition of the MAT locus flanking regions in 11 species of Ceratocystidaceae representing four genera. Genome assemblies for each species were examined to identify the MAT locus and determine the structure of the flanking regions. Large contigs containing the MAT locus were then functionally annotated and analysed for the presence of transposable elements. Genes typically flanking the MAT locus in sordariomycetes were found to be highly conserved in the Ceratocystidaceae. The different genera in the Ceratocystidaceae displayed little synteny outside of the immediate MAT locus flanking genes. Even though species ofCeratocystis did not show much synteny outside of the immediate MAT locus flanking genes, species of Huntiella and Endoconidiophora were comparatively syntenic. Due to the high number of transposable elements present in Ceratocystis MAT flanking regions, we hypothesise that Ceratocystis species may have undergone recombination in this region.
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http://dx.doi.org/10.1016/j.funbio.2018.09.003DOI Listing
December 2018

Chromium sequencing: the doors open for genomics of obligate plant pathogens.

Biotechniques 2018 11;65(5):253-257

Department of Biochemistry, Genetics & Microbiology, Tree Protection Co-operative Programme (TPCP), Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.

It is challenging to sequence and assemble genomes of obligate plant pathogens and microorganisms because of limited amounts of DNA, comparatively large genomes and high numbers of repeat regions. We sequenced the 1.2 gigabase genome of an obligate rust fungus, Austropuccinia psidii, the cause of rust on Myrtaceae, with a Chromium 10X library. This technology has mostly been applied for single-cell sequencing in immunological studies of mammals. We compared scaffolds of a genome assembled from the Chromium library with one assembled from combined paired-end and mate-pair libraries, sequenced with Illumina HiSeq. Chromium 10X provided a superior assembly, in terms of number of scaffolds, N50 and number of genes recovered. It required less DNA than other methods and was sequenced and assembled at a lower cost. Chromium sequencing could provide a solution to sequence and assemble genomes of obligate plant pathogens where the amount of available DNA is a limiting factor.
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http://dx.doi.org/10.2144/btn-2018-0019DOI Listing
November 2018

Armillaria Root-Rot Pathogens: Species Boundaries and Global Distribution.

Pathogens 2018 Oct 24;7(4). Epub 2018 Oct 24.

Department of Biochemistry, Genetics and Microbiology (BGM), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa.

This review considers current knowledge surrounding species boundaries of the Armillaria root-rot pathogens and their distribution. In addition, a phylogenetic tree using translation elongation factor subunit 1-alpha (-1α) from isolates across the globe are used to present a global phylogenetic framework for the genus. Defining species boundaries based on DNA sequence-inferred phylogenies has been a central focus of contemporary mycology. The results of such studies have in many cases resolved the biogeographic history of species, mechanisms involved in dispersal, the taxonomy of species and how certain phenotypic characteristics have evolved throughout lineage diversification. Such advances have also occurred in the case of spp. that include important causal agents of tree root rots. This commenced with the first phylogeny for that was based on IGS-1 (intergenic spacer region one) DNA sequence data, published in 1992. Since then phylogenies were produced using alternative loci, either as single gene phylogenies or based on concatenated data. Collectively these phylogenies revealed species clusters in linked to their geographic distributions and importantly species complexes that warrant further research.
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http://dx.doi.org/10.3390/pathogens7040083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6313743PMC
October 2018

Diversity and evolution of polyketide biosynthesis gene clusters in the Ceratocystidaceae.

Fungal Biol 2018 09 4;122(9):856-866. Epub 2018 May 4.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0002, South Africa. Electronic address:

Polyketides are secondary metabolites with diverse biological activities. Polyketide synthases (PKS) are often encoded from genes clustered in the same genomic region. Functional analyses and genomic studies show that most fungi are capable of producing a repertoire of polyketides. We considered the potential of Ceratocystidaceae for producing polyketides using a comparative genomics approach. Our aims were to identify the putative polyketide biosynthesis gene clusters, to characterize them and predict the types of polyketide compounds they might produce. We used sequences from nineteen species in the genera, Ceratocystis, Endoconidiophora, Davidsoniella, Huntiella, Thielaviopsis and Bretziella, to identify and characterize PKS gene clusters, by employing a range of bioinformatics and phylogenetic tools. We showed that the genomes contained putative clusters containing a non-reducing type I PKS and a type III PKS. Phylogenetic analyses suggested that these genes were already present in the ancestor of the Ceratocystidaceae. By contrast, the various reducing type I PKS-containing clusters identified in these genomes appeared to have distinct evolutionary origins. Although one of the identified clusters potentially allows for the production of melanin, their functional characterization will undoubtedly reveal many novel and important compounds implicated in the biology of the Ceratocystidaceae.
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http://dx.doi.org/10.1016/j.funbio.2018.04.011DOI Listing
September 2018

IMA Genome-F 9: Draft genome sequence of (syn. ), two strains, , , cf and .

IMA Fungus 2018 Jun 11;9(1):199-223. Epub 2018 Jun 11.

Biotechnology and Nuclear Technology Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu 610065, Sichuan, China.

Draft genomes of the species (syn. , two strains, , , and are presented Both mating types (MAT1-1 and MAT1-2) of are included. Two strains of that produce sulfated homotyrosine echinocandin variants, FR209602, FR220897 and FR220899 are presented. The sequencing of , and cf has enabled mapping of the gene clusters encoding the chemical diversity from the echinocandin pathways, providing data that reveals the complexity of secondary metabolism in these different species. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity (in some cases), biology and toxin production of these economically important fungi.
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http://dx.doi.org/10.5598/imafungus.2018.09.01.13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048567PMC
June 2018

Mitochondrial introgression and interspecies recombination in the species complex.

IMA Fungus 2018 Jun 27;9(1):37-48. Epub 2018 Feb 27.

Department of Genetics, Biochemistry and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.

The species complex (FFSC) is an economically important monophyletic lineage in the genus Incongruence observed among mitochondrial gene trees, as well as the multiple non-orthologous copies of the internal transcribed spacer region of the ribosomal RNA genes, suggests that the origin and history of this complex likely involved interspecies gene flow. Based on this hypothesis, the mitochondrial genomes of non-conspecific species should harbour signatures of introgression or introgressive hybridization. The aim of this study was therefore to search for recombination between the mitochondrial genomes of different species in the FFSC. Using methods based on mt genome sequence similarity, five significant recombinant regions in both gene and intergenic regions were detected. Using coalescent-based methods and the sequences for individual mt genes, various ancestral recombination events between different lineages of the FFSC were also detected. These findings suggest that interspecies gene flow and introgression are likely to have played key roles in the evolution of the FFSC at both ancient and more recent time scales.
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http://dx.doi.org/10.5598/imafungus.2018.09.01.04DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048563PMC
June 2018
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