Assessing genetic diversity and virulence in Fusarium oxysporum f. sp. vasinfectum and evaluating putative secreted DNases as virulence factors

Abstract

The Fusarium oxysporum species complex consists of asexual soil-borne fungi that cause vascular diseases of various plant hosts. Fusarium oxysporum f. sp. vasinfectum (Fov) infects the xylem of cotton and other host plants. As cotton is an important crop in Alabama and the United States, and few management strategies besides host resistance are effective for controlling Fov, this research aimed to better understand the pathogenicity of Fov so that better management strategies can be developed. This research also sought to better understand a new virulent genotype of Fov in the United States known as race 4. In the first research chapter, 118 field isolates from Alabama were assessed for genetic diversity using TEF1/RPB2 phylogeny, hydroponic virulence assays, and pulsed-field gel electrophoresis. Eighteen different haplotypes were identified and karyotypes of small chromosomes varied across haplotypes, suggesting Fov isolates from Alabama are genetically diverse, but no race 4 isolates were recovered. Fov isolates from a haplotype containing race 1 isolates were unexpectedly aggressive on both Pima and Upland cotton. Fov isolates from this haplotype may be a more pressing concern in Alabama currently than race 4. The second research chapter investigates nucleic acid extracellular traps (NETs), consisting of nucleic acid, histones, and other antimicrobial peptides which may protect plant roots from infection and extracellular DNases (exDNases) in Fov. Some pathogens use exDNases to destroy NETs to allow them to infect their hosts. Manipulating the interaction of NETs produced by plant roots and pathogen exDNases may lead to new management strategies for soil-borne pathogens such as Fov. In this study, we show that two exDNases are induced upon infection of cotton plants in Fov, and mutation of one exDNase reduced virulence in Fov in hydroponic test tube assays. Complementation of the DNase gene restored this mutant to the virulence of the wild type. This shows for the first time the involvement of putative exDNases in virulence for a soil-borne fungal pathogen. The last research chapter investigates race 4 isolates and potential cotton specific genes. Race 4 isolates were found to vary in levels of virulence as well as numbers and sizes of putative accessory chromosomes. All Fov genomes besides a race 5 isolate possess homologs not present in most other Fo genomes. The region containing these homologs is over 10× the size of other genotypes in race 4 isolates and contains an abundance of repetitive elements. These twelve homologous genes may be important for virulence on cotton and may be involved in defoliation. Overall, this research found variation in virulence and putative accessory chromosomes across and within genotypes of Fov. Twelve gene homologs were identified which may be associated with cotton specific virulence and will be further investigated. exDNases were also found to be involved in virulence, and these will be further profiled in the future to identify potential mechanisms of action.