Molecular taxonomic studies of selected species in the Gibberella fujikuroi complex

Thesis (PhD)--University of Pretoria, 2006.Microbiology and Plant PathologyUnrestricte

Molecular Taxonomic Studies Of Selected Species In The Gibberella Fujikuroi Complex

Fusarium subglutinans forms part of the Gibberella fujikuroi complex. This species aggregate has been associated with different plant hosts, as well as at least three mating populations in the G. fujikuroi complex. However, F. subglutinans is a polyphyletic taxon (= F. subglutinans sensu lato ), because each of these mating populations and lineages associated with the different hosts constitutes a discrete species. These different species are virtually identical when compared using morphological characteristics. The major goal of this study was to characterize some of the species representing F. subglutinans sensu lato using various protein-coding gene sequences. Methods to aid in the identification and differentiation of the fungi comprising F. subglutinans sensu lato were also developed. This thesis is, therefore, presented as a compilation of chapters that each deals with different approaches and techniques to study the taxonomy and biology of F. subglutinans sensu lato. Most studies on the phylogeny and taxonomy of species in the G. fujikuroi complex have used protein-coding gene sequences. This is because the more widely used ribosomal DNA sequences do not provide sufficient resolution in these fungi. Their use is also complicated by the presence of paralogous internal transcribed spacer regions. Although several authors have presented extensive reviews on the use of ribosomal DNA sequences in taxonomy, no such reviews are available for the use of genes that encode proteins. A comprehensive literature review dealing with this issue is, therefore, presented in chapter one. Pitch canker of mature pines and root rot of pine seedlings are important forestry diseases. The causal agent of these diseases is F. subglutinans f. sp. pini. This fungus is morphologically almost indistinguishable from other species of F. subglutinans sensu lato. The lack of a reliable identification system has hampered the implementation of disease management programs and quarantine measures. In chapter two, a PCR-based identification technique for F. subglutinans f. sp. pini is reported. This technique is based on the presence of unique restriction fragment length polymorphisms in the amplified portion of the histone HJ gene of this fungus. The biological species concept is frequently used for studying the fungi in the G. fujikuroi complex. Application of this concept for the classification of these fungi is relatively straightforward since they are heterothallic, having one of two possible mating types (MAT-1 or MAT-2). To simplify the identification of isolates with opposite mating types, a PCR-based method was developed and is presented in chapter three. Partial DNA sequences for the two MAT loci (idiomorphs) were determined and used to construct mating type specific primers. Chapter four reports on two distinct phylogenetic species associated with the disease known 11 Digitised by the Department of Library Services in support of open access to information, University of Pretoria, 2021 as mango malformation. This study was based on the DNA sequence for the histone HJ and Ptubulin genes. Both of the species associated with mango malformation display morphological characters typical of F. subglutinans. Since reliable identification procedures have not been available, the possible identity of these species was determined using a public domain nucleotide database and the internet-based programme BLAST. Application of the biological species concept for classifying Fusarium isolates does not always yield definitive resolution of species. For example, sexually compatible isolates will be classified as the same species, whereas incompatible fungi do not necessarily represent separate species. Apart from "species barriers", factors such as female sterility and low fertility, greatly influence sexual compatibility tests. These factors may result in the genetic isolation of a population or species. Chapter five reports on such a genetically isolated population of isolates. Using the DNA sequence of two protein-coding genes, this population was shown to form part of the G.fujikuroi mating population E (F. subglutinans sensu stricto). Chapter six reports on a recent speciation event within G. fujikuroi mating population E. The two cryptic species that emerged from the study are morphologically identical. They also have similar primary hosts, i.e. Zea spp. The speciation event was detected using a phylogenetic approach. For this purpose specific regions of three nuclear protein-coding genes, as well as three nuclear regions of unknown function were used. Chapter seven represents a molecular and morphological comparison of the species comprising F. subglutinans sensu lato. All the available Fusarium isolates that were previously shown to display morphological characters that are typical of F. subglutinans were compared. From these comparisons, diagnostic morphological characters were identified. All these fungi were also compared using the gene sequences for three nuclear protein-coding genes, as well as the sequence for the mitochondrial small subunit ribosomal RNA gene. From the sequence of the most variable of these regions (elongation factor 1a), diagnostic restriction enzyme recognition sites were identified and used for species delimitation. Each of the seven chapters of this thesis represents an independent unit, three of which have already appeared in print. Studies were undertaken over a five-year period and repetition between chapters has been unavoidable. All the available synonyms for each Fusarium species are also not always provided. This was done to avoid confusion, since the current taxonomic status of many Fusarium species in the G. fujikuroi complex remains uncertain. Many of the lineages in F. subglutinans sensu lato have been renamed, whereas others have not yet been formally described. The fact that many of these newly described species are invalid (Index of fungi, 1999, 6:979-980) further complicates their nomenclature.Thesis (PhD)--University of Pretoria, 2000.Microbiology and Plant PathologyPhDUnrestricte

Extracellular vesicles in teasing apart complex plant-microbiota links : implications for microbiome-based biotechnology

Extracellular vesicles (EVs) are subcellular carriers of bioactive compounds with a complex array of functional effects on target cells. In mammals, circulating bodily fluid microbiota EVs (mbEVs) deliver cargo from source cells and adversely or favorably alter the physiology of the same source, neighboring, and distant recipient cells in an autocrine, paracrine, or endocrine fashion, respectively. Plant mbEVs may similarly mediate these interactive effects within the holobiont framework. However, the majority of plant EV research has focused on a small number of individual microbes, thus failing to reflect the importance of EVs in a community and consequently leaving a wide gap in scientific knowledge. Addressing this gap should entail a systemslevel approach that combines vesicle characterization with microbiome analyses. This would certainly usher in a new age in microbial biotechnology entailing EVs as a microbiome manipulation strategy, a biomarker for stable microbiomes, and a diagnostic tool for plant infectious diseases.https://journals.asm.org/journal/msystemspm2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Three novel lineages of 'Candidatus Liberibacter africanus' associated with native rutaceous hosts of Trioza erytreae in South Africa

Greening disease of citrus in South Africa is associated with ‘Candidatus Liberibacter africanus’ (Laf), a phloem-limited bacterium vectored by the sap-sucking insect Trioza erytreae (Triozidae). Despite the implementation of control strategies, this disease remains problematic, suggesting the existence of reservoir hosts to Laf. The current study aimed to identify such hosts. Samples from 234 trees of Clausena anisata, 289 trees of Vepris lanceolata and 231 trees of Zanthoxylum capense were collected throughout the natural distribution of these trees in South Africa. Total DNA was extracted from samples and tested for the presence of liberibacters by a generic Liberibacter TaqMan real-time PCR assay. Liberibacters present in positive samples were characterized by amplifying and sequencing rplJ, omp and 16S rRNA gene regions. The identity of tree host species from which liberibacter sequences were obtained was verified by sequencing host rbcL genes. Of the trees tested, 33 specimens of Clausena, 17 specimens of Vepris and 10 specimens of Zanthoxylum tested positive for liberibacter. None of the samples contained typical citrus-infecting Laf sequences. Phylogenetic analysis of 16S rRNA gene sequences indicated that the liberibacters obtained from Vepris and Clausena had 16S rRNA gene sequences identical to that of ‘Candidatus Liberibacter africanus subsp. capensis’ (LafC), whereas those from Zanthoxylum species grouped separately. Phylogenetic analysis of the rplJ and omp gene regions revealed unique clusters for liberibacters associated with each tree species. We propose the following names for these novel liberibacters: ‘Candidatus Liberibacter africanus subsp. clausenae’ (LafCl), ‘Candidatus Liberibacter africanus subsp. vepridis’ (LafV) and ‘Candidatus Liberibacter africanus subsp. zanthoxyli’ (LafZ). This study did not find any natural hosts of Laf associated with greening of citrus. While native citrus relatives were shown to be infected with Laf-related liberibacters, nucleotide sequence data suggest that these are not alternative sources of Laf to citrus orchards, per se.Citrus Research International (CRI), the National Research Foundation (NRF) and the Department of Science and Technology (DST)/NRF Centre of Excellence in Tree Health Biotechnology (CTHB).http://ijs.sgmjournals.orghb2016Forestry and Agricultural Biotechnology Institute (FABI)Microbiology and Plant Patholog

Polyphasic taxonomic characterization of lactic acid bacteria isolated from spontaneous sorghum fermentations used to produce ting, a traditional South African food

Ting, an indigenous cooked fermented food made from sorghum flour, is consumed extensively in South Africa. Due to the spontaneous nature of the sorghum fermentation considerable variations in the sensory and microbial quality of the end-product may occur, thus hampering large-scale production of this food. The use of starter cultures purified from the fermented sorghum may be an alternative approach to obtain ting of consistent quality. The aim of this study was therefore to identify the lactic acid bacteria (LAB) associated with ting fermentation using a polyphasic approach. Phenotypic characterization and sequence analysis of the genes encoding the 16S subunit of the ribosomal RNA (rrs) and phenylalanyl tRNA synthase (pheS) were used. The results of these analyses showed that ting fermentation involved at least three different species of LAB, i.e. Lactobacillus fermentum, L. plantarum and L. rhamnosus. To our knowledge, this is the first report of polyphasic taxonomic characterization of LAB from this food. This research forms an essential first step towards the development of relevant starter cultures to produce ting of consistent quality

Phylogenomic analyses predict sistergroup relationship of nucleariids and Fungi and paraphyly of zygomycetes with significant support

Abstract Background Resolving the evolutionary relationships among Fungi remains challenging because of their highly variable evolutionary rates, and lack of a close phylogenetic outgroup. Nucleariida, an enigmatic group of amoeboids, have been proposed to emerge close to the fungal-metazoan divergence and might fulfill this role. Yet, published phylogenies with up to five genes are without compelling statistical support, and genome-level data should be used to resolve this question with confidence. Results Our analyses with nuclear (118 proteins) and mitochondrial (13 proteins) data now robustly associate Nucleariida and Fungi as neighbors, an assemblage that we term 'Holomycota'. With Nucleariida as an outgroup, we revisit unresolved deep fungal relationships. Conclusion Our phylogenomic analysis provides significant support for the paraphyly of the traditional taxon Zygomycota, and contradicts a recent proposal to include Mortierella in a phylum Mucoromycotina. We further question the introduction of separate phyla for Glomeromycota and Blastocladiomycota, whose phylogenetic positions relative to other phyla remain unresolved even with genome-level datasets. Our results motivate broad sampling of additional genome sequences from these phyla

Relevance of prokaryotic subspecies in the age of genomics

The availability of multiple gene sequences, and in particular full genome sequence data, for microbial strains has changed how taxonomists delineate subspecies belonging to the Archaea and Bacteria. Well-defined phylogenetic lineages that share higher genome similarity values compared to the widely used species thresholds are often described as subspecies, despite clear evidence of genetic isolation between them. These well-defined lineages, reflecting notable genetic isolation of the core genome represent more recently evolved, unique and sui generis evolutionary units. Because they bear all of the hallmarks of species, most contemporary subspecies likely represent species in their own right. Although there is considerable value in defining intraspecies variation (e.g., pathovar, serovar and symbiovar), the discriminating properties of such units are mostly encoded on accessory subgenomic compartments. We therefore argue that the taxonomic category of subspecies has become irrelevant and propose that its use should be discontinued. This will minimize inconsistencies related to the subjective nature of species-subspecies distinctions. Formal recognition of biologically relevant variation within species based on the accessory genome information will have practical significance in fields such as clinical, industrial and agricultural microbiology.https://www.journals.elsevier.com/new-microbes-and-new-infectionshj2023BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Legume nodules from nutrient-poor soils exhibit high plasticity of cellular phosphorus recycling and conservation during variable phosphorus supply

Nitrogen fixing legumes rely on phosphorus for nodule formation, nodule function and the energy costs of fixation. Phosphorus is however very limited in soils, especially in ancient sandstone-derived soils such as those in the Cape Floristic Region of South Africa. Plants growing in such areas have evolved the ability to tolerate phosphorus stress by eliciting an array of physiological and biochemical responses. In this study we investigated the effects of phosphorus limitation on N2 fixation and phosphorus recycling in the nodules of Virgilia divaricata (Adamson), a legume native to the Cape Floristic Region. In particular, we focused on nutrient acquisition efficiencies, phosphorus fractions and the exudation and accumulation of phosphatases. Our finding indicate that during low phosphorus supply, V. divaricata internally recycles phosphorus and has a lower uptake rate of phosphorus, as well as lower levels adenylates but greater levels of phosphohydrolase exudation suggesting it engages in recycling internal nodule phosphorus pools and making use of alternate bypass routes in order to conserve phosphorus.DST-NRF Centre of Excellence in TreeHealth Biotechnology (CTHB).http://www.elsevier.com/locate/jplph2017-02-28hb201