Molecular Taxonomic Studies Of Selected Species In The Gibberella Fujikuroi Complex

Abstract

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.