Gerda Fourie

Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups

ABSTRACT Fusarium oxysporum f. sp. cubense, the causal agent of fusarium wilt of banana (Musa spp.), is one of the most destructive strains of the vascular wilt fungus F. oxysporum. Genetic relatedness among and within vegetative compatibility groups (VCGs) of F. oxysporum f. sp. cubense was studied by sequencing two nuclear and two mitochondrial DNA regions in a collection of 70 F. oxysporum isolates that include representatives of 20 VCGs of F. oxysporum f. sp. cubense, other formae speciales, and nonpathogens. To determine the ability of F. oxysporum f. sp. cubense to sexually recombine, crosses were made between isolates of opposite mating types. Phylogenetic analysis separated the F. oxysporum isolates into two clades and eight lineages. Phylogenetic relationships between F. oxysporum f. sp. cubense and other formae speciales of F. oxysporum and the relationships among VCGs and races of F. oxysporum f. sp. cubense clearly showed that F. oxysporum f. sp. cubenses ability to cause disease on banana has emerged multiple times, independently, and that the ability to cause disease to a specific banana cultivar is also a polyphyletic trait. These analyses further suggest that both coevolution with the host and horizontal gene transfer may have played important roles in the evolutionary history of the pathogen. All examined isolates harbored one of the two mating-type idiomorphs, but never both, which suggests a heterothallic mating system should sexual reproduction occur. Although, no sexual structures were observed, some lineages of F. oxysporum f. sp. cubense harbored MAT-1 and MAT-2 isolates, suggesting a potential that these lineages have a sexual origin that might be more recent than initially anticipated.

Current status of the taxonomic position of Fusarium oxysporum formae specialis cubense within the Fusarium oxysporum complex

Fusarium oxysporum is an asexual fungal species that includes human and animal pathogens and a diverse range of nonpathogens. Pathogenic and nonpathogenic strains of this species can be distinguished from each other with pathogenicity tests, but not with morphological analysis or sexual compatibility studies. Substantial genetic diversity among isolates has led to the realization that F. oxysporum represents a complex of cryptic species. F. oxysporum f. sp cubense (Foc), causal agent of Fusarium wilt of banana, is one of the more than 150 plant pathogenic forms of F. oxysporum. Multi-gene phylogenetic studies of Foc revealed at least eight phylogenetic lineages, a finding that was supported by random amplified polymorphic DNAs, restriction fragment length polymorphisms and amplified fragment length polymorphisms. Most of these lineages consist of isolates in closely related vegetative compatibility groups, some of which possess opposite mating type alleles, MAT-1 and MAT-2; thus, the evolutionary history of this fungus may have included recent sexual reproduction. The ability to cause disease on all or some of the current race differential cultivars has evolved convergently in the taxon, as members of some races appear in different phylogenetic lineages. Therefore, various factors including co-evolution the plant host and horizontal gene transfer are thought to have shaped the evolutionary history of Foc. This review discusses the evolution of Foc as a model formae specialis in F. oxysporum in relation to recent research findings involving DNA-based studies.

IMA Genome-F 4: Draft genome sequences of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygamai, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata.

The genomes of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygami, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata are presented in this genome announcement. These seven genomes are from endophytes, plant pathogens and economically important fungal species. The genome sizes range from 26.6 Mb in the case of Leptographium lundbergii to 44 Mb for Chrysoporthe austroafricana. The availability of these genome data will provide opportunities to resolve longstanding questions regarding the taxonomy of species in these genera, and may contribute to our understanding of the lifestyles through comparative studies with closely related organisms.

Association of the pitch canker pathogen Fusarium circinatum with grass hosts in commercial pine production areas of South Africa

The pitch canker pathogen, Fusarium circinatum, has major impacts on production in pine nurseries and plantations in South Africa. Thus far, efforts to reduce local spread have focused on rouging of infected pines and sanitation to eliminate local sources of inoculum. Although the host range of F. circinatum was thought to be limited to pines and Douglas-fir, recent studies in California indicate that this fungus is capable of infecting grasses as a symptomless endophyte. Consequently, it is possible that grasses represent a reservoir of inoculum that influences the occurrence of disease in South African pine nurseries and plantations. The objectives of this study were to survey a wide range of grass species in both nurseries and plantations in South Africa for the presence of F. circinatum. In all, 22 species of grass were sampled at a nursery in Mpumulanga and in a plantation on the Western Cape. Isolates obtained from grasses were identified based on morphological criteria and DNA sequence data. Fusarium circinatum was recovered from vegetative tissues of four grass species including Briza maxima, Ehrharta erecta var. erecta, Pentameris pallida and one species that could not be identified. All isolates were pathogenic to pines and comparable in virulence to a known F. circinatum isolate that was included as a positive control. These studies indicate that grasses may constitute inoculum reservoirs that could facilitate persistence and dissemination of the pathogen in nurseries, and provide a means for the pathogen to move between widely separated pine stands, where grass hosts occur in intervening areas.

Evidence for inter-specific recombination among the mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex

BackgroundThe availability of mitochondrial genomes has allowed for the resolution of numerous questions regarding the evolutionary history of fungi and other eukaryotes. In the Gibberella fujikuroi species complex, the exact relationships among the so-called “African”, “Asian” and “American” Clades remain largely unresolved, irrespective of the markers employed. In this study, we considered the feasibility of using mitochondrial genes to infer the phylogenetic relationships among Fusarium species in this complex. The mitochondrial genomes of representatives of the three Clades (Fusarium circinatum, F. verticillioides and F. fujikuroi) were characterized and we determined whether or not the mitochondrial genomes of these fungi have value in resolving the higher level evolutionary relationships in the complex.ResultsOverall, the mitochondrial genomes of the three species displayed a high degree of synteny, with all the genes (protein coding genes, unique ORFs, ribosomal RNA and tRNA genes) in identical order and orientation, as well as introns that share similar positions within genes. The intergenic regions and introns generally contributed significantly to the size differences and diversity observed among these genomes. Phylogenetic analysis of the concatenated protein-coding dataset separated members of the Gibberella fujikuroi complex from other Fusarium species and suggested that F. fujikuroi (“Asian” Clade) is basal in the complex. However, individual mitochondrial gene trees were largely incongruent with one another and with the concatenated gene tree, because six distinct phylogenetic trees were recovered from the various single gene datasets.ConclusionThe mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex are remarkably similar to those of the previously characterized Fusarium species and Sordariomycetes. Despite apparently representing a single replicative unit, all of the genes encoded on the mitochondrial genomes of these fungi do not share the same evolutionary history. This incongruence could be due to biased selection on some genes or recombination among mitochondrial genomes. The results thus suggest that the use of individual mitochondrial genes for phylogenetic inference could mask the true relationships between species in this complex.

The distribution and host range of the banana fusarium wilt fungus, fusarium oxysporum f. sp. cubense , in Asia

Fusarium oxysporum formae specialis cubense (Foc) is a soil-borne fungus that causes Fusarium wilt, which is considered to be the most destructive disease of bananas. The fungus is believed to have evolved with its host in the Indo-Malayan region, and from there it was spread to other banana-growing areas with infected planting material. The diversity and distribution of Foc in Asia was investigated. A total of 594 F. oxysporum isolates collected in ten Asian countries were identified by vegetative compatibility groups (VCGs) analysis. To simplify the identification process, the isolates were first divided into DNA lineages using PCR-RFLP analysis. Six lineages and 14 VCGs, representing three Foc races, were identified in this study. The VCG complex 0124/5 was most common in the Indian subcontinent, Vietnam and Cambodia; whereas the VCG complex 01213/16 dominated in the rest of Asia. Sixty-nine F. oxysporum isolates in this study did not match any of the known VCG tester strains. In this study, Foc VCG diversity in Bangladesh, Cambodia and Sri Lanka was determined for the first time and VCGs 01221 and 01222 were first reported from Cambodia and Vietnam. New associations of Foc VCGs and banana cultivars were recorded in all the countries where the fungus was collected. Information obtained in this study could help Asian countries to develop and implement regulatory measures to prevent the incursion of Foc into areas where it does not yet occur. It could also facilitate the deployment of disease resistant banana varieties in infested areas.

Culture-independent detection and quantification of Fusarium circinatum in a pine-producing seedling nursery

The primary symptoms associated with Fusarium circinatum infection in pine seedling nurseries are root and collar rot, shoot and tip die-back and seedling mortality. Management of this pathogen in nurseries usually involves the integration of various strategies relating to sanitation, insect control and fungicide treatment. The overall goal of this study was to use quantitative real-time PCR (qPCR) to detect and quantify the airborne inoculum of F. circinatum in a commercial pine seedling nursery. For this purpose, an existing qPCR method was optimised and evaluated for its efficacy to quantify and monitor airborne conidia over a one-year period. Results showed that F. circinatum occurred at relatively low levels in the nursery throughout the year and that its distribution was spatially sporadic. The data suggest that standard nursery sanitation practices in the test nursery maintained the airborne inoculum of F. circinatum at low levels. The uneven distribution of infection also suggests that airborne inoculum does not represent the primary source of inoculum for the F. circinatum-associated seedling disease.

Genome-Wide Macrosynteny among Fusarium Species in the Gibberella fujikuroi Complex Revealed by Amplified Fragment Length Polymorphisms

The Gibberella fujikuroi complex includes many Fusarium species that cause significant losses in yield and quality of agricultural and forestry crops. Due to their economic importance, whole-genome sequence information has rapidly become available for species including Fusarium circinatum, Fusarium fujikuroi and Fusarium verticillioides, each of which represent one of the three main clades known in this complex. However, no previous studies have explored the genomic commonalities and differences among these fungi. In this study, a previously completed genetic linkage map for an interspecific cross between Fusarium temperatum and F. circinatum, together with genomic sequence data, was utilized to consider the level of synteny between the three Fusarium genomes. Regions that are homologous amongst the Fusarium genomes examined were identified using in silico and pyrosequenced amplified fragment length polymorphism (AFLP) fragment analyses. Homology was determined using BLAST analysis of the sequences, with 777 homologous regions aligned to F. fujikuroi and F. verticillioides. This also made it possible to assign the linkage groups from the interspecific cross to their corresponding chromosomes in F. verticillioides and F. fujikuroi, as well as to assign two previously unmapped supercontigs of F. verticillioides to probable chromosomal locations. We further found evidence of a reciprocal translocation between the distal ends of chromosome 8 and 11, which apparently originated before the divergence of F. circinatum and F. temperatum. Overall, a remarkable level of macrosynteny was observed among the three Fusarium genomes, when comparing AFLP fragments. This study not only demonstrates how in silico AFLPs can aid in the integration of a genetic linkage map to the physical genome, but it also highlights the benefits of using this tool to study genomic synteny and architecture.

Effect on nursery and field performance of Pinus patula seedlings after inoculation with Fusarium circinatum

Fusarium circinatum is an important fungal pathogen of Pinus species. In South Africa, it is the most significant pathogen of Pinus patula seedlings in forestry nurseries where it presents a substantial constraint to productivity and can continue to cause mortality in-field for up to two years after establishment. This study describes the results from two trials where P. patula seedlings were inoculated with F. circinatum to determine the impact of the pathogen on nursery and field performance. Seedlings were also subjected to water stress treatments to ascertain whether this would trigger the onset of disease symptoms. Inoculum load and timing of inoculation had significant effects on seedling survival in both the nursery and field. High inoculum concentrations caused greater levels of mortality and, where seedlings were inoculated at a young age, they showed higher levels of susceptibility to F. circinatum. Temporary water-stress in the nursery produced smaller plants and improved in-field survival, but this treatment did not trigger higher mortality in inoculated treatments. On the other hand, transplant stress was a major contributor to the higher levels of mortality observed in inoculated treatments. Overall, these studies confirmed that infection in the nursery leads to the disease problems observed during early plant establishment in the field.

Re-use of seedling containers and Fusarium circinatum association with asymptomatic Pinus patula planting stock

Fusarium circinatum is a pathogen causing serious post-planting mortality of Pinus patula seedlings in southern Africa. Containerised planting stock that is asymptomatic but associated with F. circinatum in the nursery is thought to be the cause of this problem. The aim of this study was to determine if re-use of seedling containers could be a source of inoculum resulting in asymptomatic planting stock and increased post-planting mortality of P. patula. Two experiments were conducted in successive years comparing nursery cull of symptomatic seedlings, seedling growth, association of F. circinatum with asymptomatic seedlings and post-planting mortality for crops raised in re-used containers, with and without sanitation, and factory-new containers. Each experiment consisted of a nursery production trial followed by out-planting into pots to assess post-planting mortality. Our results show that re-use of containers without sanitation increases the cull of symptomatic seedlings, incidence of F. circinatum associated with asymptomatic seedlings and post-planting mortality compared with the re-use of containers after steam sanitation or factory-new containers. Growth of asymptomatic seedlings was unaffected by container treatment or association with F. circinatum and in the absence of wilt symptoms the root system did not exhibit typical discolouration. Watering frequency did not influence post-planting mortality in pots. The comparison of two open-pollinated seed mixes of P. patula that, based on seedling stem inoculation screening, represented susceptible and tolerant material did not show differences in nursery cull or post-planting mortality. This work demonstrated that natural contamination of re-used containers can be a primary source of inoculum producing asymptomatic seedlings associated with F. circinatum that will succumb to the pathogen after field planting. The process of seedling infection, apparent latent infection in the seedling and expression of disease after planting needs greater understanding to improve nursery hygiene measures to control this disease.