Fusarium verticillioides is one of the most important fungal pathogens of maize and has also received increasing attention due to its ability to produce various secondary metabolites, including fumonisin B1 (FB1) and bikaverin. However, little is known about the regulatory mechanisms associated with F. verticillioides secondary metabolism. In this study, I utilized functional genomics, forward and reverse genetics, proteomics, and high efficiency homologous recombination, to better understand the complex secondary metabolism regulations in F. verticillioides. First, using the reverse genetics approach, I characterized a putative protein phosphatase gene, CPP1 as a negative regulator of FB1 biosynthesis. CPP1 gene deletion also affected multiple phenotypes such as radial growth, conidia germination rates, macroconidia formation, and hyphal swelling. Through gene complementation, I also demonstrated that the F. verticillioides CPP1 and Neurospora crassa wild-type ppe-1 gene are functionally conserved. Second, I used proteomics and quantitative real-time (qRT)-PCR, to advance our understanding of genes associated with fumonisin production. I analyzed the proteomic changes associated with the mutation in FCC1, a key positive regulator of fumonisins biosynthesis. I isolated proteins that were significantly up-regulated in either the wild-type or the fcc1 mutant, and transcriptional profiles of the genes corresponding to the selected proteins were analyzed via qRT-PCR. These genes showing expression patterns concomitant with fumonisin biosynthesis can be identified as primary targets for functional analysis. Next, I utilized REMI (Restriction Enzyme Mediated Integration) to isolate GAC1 gene, which encodes a GTPase activating protein, that serve as a negative regulator of bikaverin biosynthesis in F. verticillioides. AREA and PKS4 are downstream genes that are regulated positively and negatively by GAC1, respectively. Lastly, I generated a highly efficient homologous recombination strain of F. verticillioides. In eukaryotes, KU70 and KU80 play important roles in nonhomologous end-joining process, which leads to a high percentage of ectopic integration events during fungal transformation. By generating a KU70 gene deletion mutant (SF41), I have established a resource that will contribute to functional genomic research in F. verticillioides.

Fusarium verticillioides is one of the most important fungal pathogens of maize and has also received increasing attention due to its ability to produce various secondary metabolites, including fumonisin B1 (FB1) and bikaverin. However, little is known about the regulatory mechanisms associated with F. verticillioides secondary metabolism. In this study, I utilized functional genomics, forward and reverse genetics, proteomics, and high efficiency homologous recombination, to better understand the complex secondary metabolism regulations in F. verticillioides. First, using the reverse genetics approach, I characterized a putative protein phosphatase gene, CPP1 as a negative regulator of FB1 biosynthesis. CPP1 gene deletion also affected multiple phenotypes such as radial growth, conidia germination rates, macroconidia formation, and hyphal swelling. Through gene complementation, I also demonstrated that the F. verticillioides CPP1 and Neurospora crassa wild-type ppe-1 gene are functionally conserved. Second, I used proteomics and quantitative real-time (qRT)-PCR, to advance our understanding of genes associated with fumonisin production. I analyzed the proteomic changes associated with the mutation in FCC1, a key positive regulator of fumonisins biosynthesis. I isolated proteins that were significantly up-regulated in either the wild-type or the fcc1 mutant, and transcriptional profiles of the genes corresponding to the selected proteins were analyzed via qRT-PCR. These genes showing expression patterns concomitant with fumonisin biosynthesis can be identified as primary targets for functional analysis. Next, I utilized REMI (Restriction Enzyme Mediated Integration) to isolate GAC1 gene, which encodes a GTPase activating protein, that serve as a negative regulator of bikaverin biosynthesis in F. verticillioides. AREA and PKS4 are downstream genes that are regulated positively and negatively by GAC1, respectively. Lastly, I generated a highly efficient homologous recombination strain of F. verticillioides. In eukaryotes, KU70 and KU80 play important roles in nonhomologous end-joining process, which leads to a high percentage of ectopic integration events during fungal transformation. By generating a KU70 gene deletion mutant (SF41), I have established a resource that will contribute to functional genomic research in F. verticillioides.

The ascomycete Fusarium verticillioides (Sacc.) Nirenberg (teleomorph: Gibberella moniliformis Wineland) causes stalk and ear rots on maize worldwide. In addition to the economic losses due to reduced yield, the fungus produces fumonisins on infected corn. One of the unanswered questions in mycotoxin research is how fungi perceive and respond to various extracellular stimuli and produce mycotoxins. To date, extensive research has been performed on important signaling pathways that regulate mycotoxin biosynthesis, but little is known about the downstream target genes, notably transcription factors (TFs). While the roles of TFs have shown to be critical in eukaryotic transcription regulation, only a few have been characterized in F. verticillioides. TFs with zinc fingers have been reported in all living organisms, and in fungal species, members of the Cys2-Hys2 (C2H2) zinc finger TF family are predicted to be involved in cell differentiation, carbon utilization, and development. Using the available genomic resources, I constructed a library of C2H2 TF deletion mutants, and identified SDA1, FvFLBC and CHT1 genes with a potential role in carbon utilization, development and fumonisin B1 (FB1) biosynthesis. The Δsda1 strain showed complete growth inhibition when using sorbitol as the sole carbon source and produced higher levels of FB1 when grown on corn kernels. In addition, the Δsda1 strain produced less number of conidia compared to the wild-type progenitor. Through gene complementation, I also demonstrated that F. verticillioides SDA1 and Trichoderma reesei ACE1 are functionally conserved. FvFLBC acts as a regulator of asexual development but not FB1 biosynthesis. I also discovered that the FvFlbC N-terminus is critical for conidia production. CHT1 is associated with asexual development, fumonisin biosynthesis and pigmentation. Characterization of key signal transduction pathways, and more importantly the function of SDA1, FvFLBC and CHT1, should facilitate the elucidation of the mechanisms and regulations of growth, development, and secondary metabolism in F. verticillioides. The outcome of this study may help us determine how to minimize F. verticillioides contamination of crops and the resulting mycotoxins, providing safer and higher value corn in the US and worldwide.

Thesis (MSc)--Stellenbosch University, 2015 / ENGLISH ABSTRACT: Maize (Zea mays L.) is an important crop worldwide and forms the staple diet of many African countries including South Africa. Fusarium ear rot (FER) of maize is caused by a fungus, Fusarium verticillioides, which also produces the fumonisin mycotoxin group. The consumption of fumonisin contaminated maize grain has been associated with serious human and animal health complications. Several South African maize inbred lines exhibiting resistance to FER and fumonisin contamination have been identified. These locally adapted inbred lines could be used to generate mapping populations to identify QTLs associated with resistance to FER and fumonisin contamination. The corresponding markers could be utilised in breeding programmes through marker-assisted selection to ensure the development of commercial cultivars with resistance to FER and fumonisin contamination. In this study, resistant and susceptible maize inbred lines were utilised to commence the development of recombinant inbred line (RIL) populations for the mapping and validation of QTLs associated with FER and fumonisin resistance. One F2 population was phenotypically and genotypically analysed to produce a linkage map for the preliminary identification of QTLs associated with resistance to F. verticillioides infection and fumonisin deposition. A potential QTL for resistance to FER was detected and should be validated across several locations and years in the subsequent RIL population. Additionally, potential resistance barriers of maize to infection by F. verticillioides were investigated by histological studies. The importance of a closed stylar canal in determining resistance to FER was established for nine South African maize inbred lines by means of scanning electron microscopy (SEM). No significant association was observed between a closed stylar canal and the resistance/susceptible status of maize inbred lines before pollination, while the canals appeared closed in all inbred lines following pollination. The results suggest that the stylar canal architecture is not an essential factor determining resistance to F. verticillioides ingress in the maize inbred lines selected for this study. Furthermore, the possibility of resistance to FER and fumonisin contamination being initiated during the seedlings phase of a resistant and susceptible maize inbred line was investigated by means of confocal laser scanning microscopy (CLSM). Fusarium verticillioides growth originating from soil-borne or seed-borne contamination was monitored in various above and below soil plant tissues but no significant difference in the colonisation could be determined between resistant and susceptible maize seedlings. No fumonisin was produced regardless of the inoculation method or resistance status of the plant. These results suggests that the resistant and susceptible maize seedlings used in this study may not be resistant to systemic fungal ingress but may resist the deposition of fumonisins. The resistance associated with the resistant inbred line is not mediated during the seedling phase but potentially through structural and biochemical defence mechanisms during later plant developmental stages. / AFRIKAANSE OPSOMMING: Mielies (Zea mays L.) is ‘n belangrike graangewas wat wêreldwyd geproduseer word en dien as stapelvoedsel in talle Afrika-lande, insluitend Suid-Afrika. Fusarium kopvrot (FKV) in mielies word veroorsaak deur die swam, Fusarium verticillioides, wat ook die fumonisien mikotoksien groepe produseer. Die inname van fumonisien-geïnfekteerde mielies gaan gepaard met ernstige gesondheidsprobleme in mense en diere. Verskeie Suid-Afrikaanse ingeteelde mielielyne, wat weerstandbiedend is teen FKV en fumonisien kontaminasie, is voorheen identifiseer. Hierdie plaaslik-aangepaste teellyne kan gebruik word om kartering populasies te genereer om kwantitatiewe eienskap loci (KEL) te identifiseer wat verband hou met weerstandbiedenheid teen FKV en fumonisien kontaminasie. Die ooreenstemmende merkers kan gebruik word in teelprogramme deur gebruik te maak van merker-geassisteerde seleksie om kommersieële kultivars, wat weerstandbiedend is teenoor FKV en fumonisien kontaminasie, te ontwikkel. In hierdie studie is weerstandbiedende en vatbare mielie inteellyne gebruik om rekombinante inteellyn (RIL) populasies te begin ontwikkel vir die kartering en validasie van KEL’e geassosieer met FKV en fumonisien weerstandbiedenheid. Een F2 populasie was fenotipies en genotipies geanaliseer om ‘n koppeling-kaart te verwek vir die voorlopige identifikasie van KEL’e geassosieer met weerstandigheid tot F. verticillioides infeksie en fumonisein afsetting. ‘n Potensiële KEL vir weerstandbiedenheid is geïdentifiseer, wat verdere bevestiging in die daaropvolgende RIL populasie in verskeie geografiese areas en oor addisionele seisoene, benodig. Potensiële fisiese versperrings teen F. verticillioides tydens mieliesaad infeksie is ook ondersoek met behulp van histologiese studies. Die belangrikheid van ‘n geslote styl-kanaal vir weerstandbiedendheid teenoor FKV is bevestig in nege Suid-Afrikaanse inteellyne deur middel van skandeer elektron mikroskopie (SEM). Geen beduidende verwandskap tussen ‘n geslote styl-kanaal en die weerstandbiedenheid/vatbaarheid van die inteellyne voor bestuiwing is gevind nie, terwyl die kanaal in alle inteellyne gesluit was na bestuiwing. Die resultate dui daarop dat die styl-kanaal argitektuur nie ‘n noodsaaklike faktor is in die bepaling van weestand tot F. verticillioides besmetting in die suiwer mielielyne wat geselekteer was in hierdie studie nie. Verder is die moontlikheid dat weestand tot FKV en fumonisien kontaminasie geïnisieer kan word gedurende die saailing-fase ondersoek in beide ‘n weerstandbiedende en vatbare mielie inteellyn met behulp van konfokale laser skandering mikroskopie (CLSM). Die groei van F. verticillioides afkomstig vanuit die grond of saad is gemonitor in verskeie bo- en ondergrondse plantweefsels, maar geen beduidende verskille in kolonisasie kon opgespoor word tussen weerstandbiedende en vatbare mielie saailinge nie. Geen fumonisien produksie is waargeneem nie, ongeag die innokulasie metode of weerstand-status van die plant. Hierdie resultate dui daarop dat die weerstandbiedende en vatbare mielie saailinge wat in hierdie studie gebruik is moontlik nie weerstandbiedend is teen sistemiese swaminfeksie nie, maar wel weerstand kan bied tot afsetting van fumonisiene. Die weerstand geassosieër met die weerstandbiedende inteellyn word nie bemiddel gedurende die saailingfase nie maar waarskynlik deur strukturele en biochemiese verdedigingsmeganismes tydens latere plant ontwikkelings-stadia. / National Research Foundation (NRF)

Fusarium verticillioides is a pathogen of maize causing ear rot and stalk rot. The fungus also produces fumonisins, a group of mycotoxins linked to disorders in animals and humans. A cluster of genes, designated FUM genes, plays a key role in the synthesis of fumonisins. However, our understanding of the regulatory mechanism of fumonisin biosynthesis is limited. It was previously demonstrated that Cpp1, a protein phosphatase type 2A (PP2A) catalytic subunit, negatively regulates fumonisin production and is involved in cell shape maintenance. Typically, a structural A subunit, a catalytic C subunit, and a regulatory B subunit form PP2A heterotrimer complex. Significantly, there are two PP2A regulatory subunits in F. verticillioides genome, Ppr1 and Ppr2, which are homologous to Saccharomyces cerevisiae Cdc55 and Rts1, respectively. Based on preliminary data, I hypothesized that Ppr1 and Ppr2 are independently involved in the regulation of fumonisin biosynthesis and/or cell development, and to test this hypothesis I generated gene-deletion mutants of PPR1 and PPR2. The ppr1 deletion strain (Δppr1) resulted in drastic growth defect, but with increased microconidia production. The ppr2 deletion mutant strain (Δppr2) showed elevated fumonisin production similar to the Δcpp1 strain. Germinating Δppr1 conidia formed abnormally swollen cell with central septation. Δppr2 showed early hyphal branching during conidia germination. Results from this study suggest that two PP2A regulatory subunits in F. verticillioides carry out unique roles in regulating fumonisin biosynthesis and fungal development.

Fusarium verticillioides (Teleomorph Giberella moniliformis) is an ascomycete fungus responsible for ear and stalk rots of maize. Most importantly, it produces a group of mycotoxins called fumonisins upon colonization of maize kernels. Fumonisin B1 (FB1), the most prevalent fumonisin in nature, was first identified in 1988 and has been found to be toxic to human and animals. The gene cluster for FB1 biosynthesis and some environmental conditions responsible for the toxin production are known, but gaps in our understanding of the signaling pathways leading to FB1 biosynthesis still remain. MADS-box transcription factors (TF) are known to regulate diverse cellular functions in all eukaryotes, and in silico analyses revealed two genes, MADS1 and MAD2, in F. verticillioides. Reverse genetics studies indicated that MADS1 and MADS2 positively regulate sexual mating and FB1 biosynthesis but not pathogenicity in F. verticillioides. Furthermore, MADS1 was found to act as a broad regulator of polyketide-derived secondary metabolism. Additionally, population diversity studies were conducted in 164 F. verticillioides cultures isolated from 65 maize-producing counties in Texas. The result showed a fluid population with no particular niches formed. F. verticillioides strains were also isolated from counties that have previously tested negative for FB1 contamination in maize. The presence of the pathogen represents a risk for future FB1 contamination events if suitable conditions were to arise. My research revealed new genetic components involved in F. verticillioides secondary metabolite biosynthesis and provided a better understanding of the pathogen population fluidity in Texas.

>Magister Scientiae - MSc / Fi1sarium verticillioides is a very important mycotoxin-produeing fungus associated with maize. Fverticillioides produces a group of mycotoxins known as fumonisins under suitable environmental conditions. A series of studies was designed to provide information regarding some of the factors associated with the production offumonisin B1 (FB1) in maize patties and MYRO liquid medium. Our investigation together with previous studies have detailed the important influence of several factors on the production of fumonisins by F verticillioides strains. To understand why these strains are able to produce these toxins, an investigation into the complex interaction that occurs between biotic and abiotic parameters and their impact on fumonisin production was necessary. The results reflect the interacting factors and the intraspecific differences between strains, which may also be present in field conditions. The parameters that were varied under a predetermined set of culture conditions, included initial moisture content of maize patty cultures, temperature, initial pH and the addition of the fumonisin precursors, L-alanine and L-methionineto the cultures. Investigations into the three-way interactions of initial maize patty moisture content (30 ml water to 30g of maize), L-methionine (0.3 %) and temperature (25°C), resulted in the highest yield ofFB1 (5777.26 μgig) produced by MRC 4316. In contrast, MRC 826 was negatively affected, producing lower levels ofFB1 (3492.24 μg/g), compared to MRC 4316 at an initial moisture content (20 ml water to 30 g maize), L-methionine (0.3 %) and 25 °C. An American strain of F verticillioides MRC 7424 (= NRRL 13616), produced the highest levels of FB, (116 μg/ml), while the South African isolates, MRC 4316 and MRC 826, produced lower FB1 levels (93 and 62 μg/ml, respectively) in MYRO liquid medium. In general, FB1 production in maize patty cultures far exceeded levels obtained in liquid shake cultures. It appears that not only the ability of a particular strain of F. verticillioides, but the interaction of a variety of physiological and nutritional factors and the culture medium, are important in the production of FB,. Thus, variation of a single factor such as temperature under field conditions due to seasonal change, may therefore have a major effect on fomonisin production. A chain reaction may occur when changes in moisture, pH, etc. take place, which may influence fumonisin production further. Lyophilisation of fungal cultures proves to be an excellent method to preserve a wide range of fungi over long periods of time. It is, however, necessary to determine the viability of conidia stored in lyophilised vials at 4 ° Con a regular basis. At present, plate count methods remain the most valid technique for the detection of the viability of lyophilised conidia. Membrane-permeant nucleic acid-binding dyes (FUN-I) are viability stains that are relatively new flourescent probes for assessing the viability of metabolically active yeast cells. The purpose of this study was to microscopically determine the viability oflyophilised conidia of Fusarium and A lternaria species, using the yeast, Saccharomyces cerevisiae, as a control. FUN-1 viability stain was compared to two other staining methods, i.e. ethidium bromide (EB) and methylene blue (MB) and the viability of the conidia was compared to colony-forming units (CFU) on solid media as a control. For the purpose of determining or screening for percentage viability in a specific inoculum, results indicate that EB can be used in the case of lyophilised conidia, and MB in the case of freshly harvested conidia. Although FUN-I are recommended as a good way to determine the cell viability of a fungus, it needs relatively complicated procedures and has a time limit in which the stain can be used. The result of this study emphasize that the use of dyes to determine viability of lyophilised conidia require a critical definition of protocols for a specific fungal species, and that a good correlation with CFU needs to be demonstrated. The findings of this study could find useful applications in various studies on living and dead conidial populations. The diverse toxicological effects of fumonisins m animals and plants raised the possibility that fumonisins may also inhibit the growth of filamentous fungi. This study investigated the antifungal activity of FB1 to some h1sariu111 and other fungal species. The sensitivity of these fungi was tested by an agar-diffusion method on PDA plates. FB1 inhibited the myceliaJ growth of five of the nine fungi tested. The FB1-producing Fusarium species isolated from maize, i.e. F verticil/ioides, F glohosum and F proliferatum were resistant to FB1 even though a small inhibition zone at the highest FB1 concentration of 40mM was noted in the case of F. proliferatum. However, amongst two non-producing Fusarium spp. also isolated from maize, one (F subglutinans) was resistant and one (F graminearum) was sensitive. The most sensitive fungi tested were non-producing species not isolated from maize, i.e. A lternaria alternata, Botrytis cinerea and Penicillium expansum. The minimum inhibitory concentration ofFB1 ranged between 0.25-0.SmM for A. alternata, 1-SmM for P. expansum and B. cinerea and 5-1 OmM for F. graminearum, while the other fungi tested showed no sensitivity to FB1. This is the first report on the antifungal activity ofFB1 to filamentous fungi. Another study investigated the effect of FB1 on the germination of freshly harvested conidia of Fusarium and some other fungal species. The FB1 -producing F'usarium species isolated from maize, i. e. F vertici llioides, F. globosum and F. prolifer alum showed a decrease in germ tube length with an increase in FB1 concentrations. This indicated that these fungi can tolerate their own toxic metabolite to a ce11ain extent. However, amongst the two non-fumonisin producing Fi1sarium spp. examined, i.e. F. subglutinans and F. graminearum, isolated from maize, F. subglutinans was induced to genninate faster in the presence ofFB1 but soon developed stunted germ tubes, while F graminearum developed shorter germ tubes compared to the control cultures. The most sensitive fungi tested were species not isolated from maize, i.e. A. alternata, B. cinerea and P. expansum, which did not germinate at higher FB1 concentrations at all. Statistical analyses showed that the inhibiting effect of FB1 was highly significant (P <0.001). The conidial germination bioassay was more sensitive in the detection of the antifungal activity ofFB1 than the petri dish bioassay. The minimum inhibitory concentrations of FB1 for visible mycelial growth were closely comparable to those obtained from conidial germination. Results of these studies provide considerable information on the parameters affecting the production of FB1 and will be of great benefit in further studies focussing on fumonisin prodnction.

In questa tesi di dottorato sono stati indagati i punti critici legati ai fattori biotici e abiotici che possono influenzare la crescita del fungo Fusarium verticillioides, produttore di fumonisine in mais. le fumonisine sono metaboliti secondari prodotte da funghi appartenenti al genere Fusarium e sono state classificate come possibili cancerogene per l’uomo e per gli animali. Gli argomenti trattati nei vari capitoli sono stati: parametri ecologici che condizionano la crescita e l’accumulo di fumonisine nelle prime fasi post raccolta e durante lo stoccaggio; relazione che intercorre tra aw, umidita’ relativa e tipo di ibrido; controllo con mezzi chimici e biologici in campo e in vitro su F. verticillioides e A. flavus. / The aim of this work was to collect missing information about critical point related to abiotic and biotic factors that can influence the growth of Fusarium verticillioides in maize and the consequent production of fumonisins in kernels. Fumonisins are secondary metabolites reported as toxigenic in humans and animals. Issues treated are: variables influencing growth and toxin accumulation during post-harvest and storage; the relationship between aw, relative humidity and type of hybrids; chemical and biological control of F. verticillioides e A. flavus in field and in vitro.

Please read the abstract in the dissertation. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Chemistry / unrestricted

Fusarium verticillioides (= Fusarium moniliforme) a common fungal contaminant of maize throughout the world has been associated with diseases in both man and animals. The structure of the fumonisins, a family of structurally related mycotoxins isolated from cultures associated with the high incidence of human oesophageal cancer in the Transkei region in South Africa and with equine leucoencephalomalacia, a neurological disorder in horses and donkeys, has been established. The main mycotoxin, fumonisin B1 consists of the diester formed by the C(14) and C(15) hydroxyl groups of (2S,3S,5R,10R,12S,14S,15R,16R)-2- amino-12,16-dimethyleicosane-3,10,14,15-pentaol with the Si carboxy group of propane- 1,2,3-tricarboxylic acid. A comparison of the structures of the 28 known fumonisins reveals that they share a common structural motif for the C(11)-C(20) unit, and probably also the same stereochemistry for the 4 stereogenic centres present in this unit. Disconnection of the C(9)–C(10) bond in a retrosynthetic analysis of the fumonisins C20 backbone (C19 in the fumonisin C series)identifies (3S,5S,6R,7R)-3,7-dimethylundecane-1,5,6-triol as a common building block for the synthesis of any of the fumonisins. In the dissertation the retrosynthetic analysis of this 3,7-dimethylundecane-1,5,6-triol building block identifies (3S,4R,5R)-5-methylnonane-1,3,4-triol as a viable target which in turn could be derived from a simple starting material trans-4-hexen-3-one. Key reactions identified to realise the required transformations leading to the identified target included kinetic enzymatic resolution of the racemic alcohol obtained from trans-4-hexen-3-one, and a pivotal role for both the [2,3]-Wittig rearrangement and the use of Sharpless asymmetric epoxidation methodology as these reactions generated the requisite stereogenic centres present in (3S,4R,5R)-5-methylnonane-3,4-diol. In this manner a synthetic route from trans-4-hexen-3- one to (2R,3R,4R,5R,6E)-4-(benzyloxy)-2,3-epoxy-5-methylnon-6-en-1-ol using appropriate functional group transformations and protective group strategies, with complete stereochemical control, were developed in this work. Alternative strategies to overcome problems encountered during the synthesis are presented for future work. The conversion of the 4-(benzyloxy)-2,3-epoxy-5-methylnon-6-en-1-ol intermediate to the protected 5- methylnonane-1,3,4-triol target could not be carried out due to time constraints and material shortages. / Dissertation (MSc)--University of Pretoria, 2011. / Chemistry / unrestricted