published_or_final_version / Biological Sciences / Master / Master of Philosophy

This project investigated the use of new near infrared transmittance (NIT) technology for individual kernel sorting to reduce Fusarium damaged kernels (FDK) and mycotoxins in grain. There were two objectives: 1) determine the efficiency of sorting; and 2) assess how highly contaminated sorted grain fractions can be used in dietary challenges for broilers as a screen for methods to reduce mycotoxin exposure. Fusarium damaged kernels are associated with lower crude protein (CP) caused by fungal infestation during kernel development, and may contain varying concentrations of mycotoxins (e.g. deoxynivalenol; DON). The BoMill TriQ measures the NIT of limited spectra to predict CP variation among individual kernels at ~2 - 3 MT/hour. Five sources of downgraded grain attained from grain producers in Western Canada in 2013 were sorted into ten calibration fractions, each analyzed for CP, FDK and 16 common mycotoxins. From these analyses, three wheat sources were individually sorted into three test fractions: outliers (10%); high FDK (low CP; 20% of source); and low FDK (high CP; 70% of source). Four diet recombinations were produced based on increasing inclusion of the high % FDK fraction [0% (M0), 20% (M20), 40% (M40) or 60% (M60)] of each wheat source, providing increasing mycotoxin concentrations in the test diets. Productions of these diets from re-combining the FDK fractions enabled a 3 wheat source x 4 FDK level (M0, M20, M40, M60) factorial design. The 12 test diets were included at 70 (starter, 0 - 21 d) and 75% (grow/finisher, 21 - 35 d) of a basal diet. Diets were formulated to meet or exceed NRC (1994) requirements for broilers. Eight cages of five, one-day old male Ross 308 broilers were each randomly assigned to the 12 starter diets. The number of cages were reduced to three per diet at 21 d. Broiler performance were recorded for the 0 - 21 and 21 - 35 d. Apparent metabolizable energy (AME; kcal ME/kg diet) and nitrogen retention (NR; %) were determined using digestible markers and excreta collections. Five biomarkers of immune function were measured for starter and grower/finisher periods: 1) cell-mediated immune response to injection of the T cell mitogen phytohemagglutinin (PHA); 2) humoral response to immunization with bovine serum albumin (BSA) antigen; 3) relative weights of liver, spleen and bursa of Fabricius; 4) heterophil to lymphocyte (H:L) ratio; and 5) histopathology of primary and secondary immune organs. Analysis of sorting efficiency of this technology indicated that grain could be separated into 10% increments based on unique spectral ranges and their correlation to the chemical characteristics of CP. Indications were that the lowest 20% CP kernels contained increased FDK (15.4%) and DON (10.2 ppm) compared to the unsorted kernels (2.4% and 1.7 ppm). The statistical correlations between FDK, DON and CP provided the capability to produce high and low mycotoxin fractions for use in the poultry feeding trial. Analysis of growth and performance endpoints of each exposure period indicated no significant difference (P > 0.05), however AME and NR were different (P < 0.01) among treatment groups at 21 and 35 d. Analysis of immune system endpoints indicated no significant differences (P > 0.05) among treatment groups in cell-mediated (PHA; 0.32 - 0.35 % change), humoral (BSA; 0.57 - 0.64 % change) or H:L ratio (0.03 - 0.13 % change) immune responses. However, histopathological examination of the spleen (P < 0.05) at 21 d and the liver (P < 0.01) at 35 d showed increases in lymphoid aggregates and/or granulopoisis in the diet containing 8 ppm DON suggesting potential adverse effects on the immune system. Overall, the results of these studies indicate that the NIT technology has the potential to produce naturally contaminated diets with various levels of mycotoxins from a single source of grain. These naturally contaminated diets may improve our ability to evaluate models to examine the effects of mycotoxin exposures to poultry or livestock.

3 pp. / This publication provides information on the development and management of Fusarium wilt of lettuce. Topics covered include the characteristics of the plant pathogen, disease development, and disease management considerations.

This work encompasses studies on the phylogeny of F. oxysporum f. sp. lactucae, the development of a PCR-based seed assay for the detection of this fungus in seed, the potential of seed transmission of the fungus that may result in seed dissemination, and the genetic variation existing within pathogen populations. In phylogenetic analysis, the mtSSU and EF-1&amp;#945; sequences provided limited phylogenetic resolution and did not differentiate the lactucae isolates from other F. oxysporum isolates, while the IGS region resolved lactucae race 1 isolates as a monophyletic group with three other f. spp. of F. oxysporum. In all analyses, lactucae race 2 isolates comprised a separate lineage that was phylogenetically distinct. Based the IGS, PCR primers were designed for detection of the fungus, and a PCR-based seed assay was developed for detection of the fungus in seed. This assay allowed for detection of the pathogen from artificially infested seed lots with infestation rates as low as 0.5%. To investigate seedborne transmission, the moderately resistant cultivars Sharpshooter, Vulcan, and King Henry were inoculated and grown to maturity in the greenhouse. The pathogen was recovered from sections of surface disinfested inflorescence stalks at rates of 14.3 - 62.7% but not from the floral parts. The incidence of recovery from nondisinfested seeds was between 0.02% and 0.08%. The pathogen was not isolated from surface disinfested seeds suggesting that it was externally seedborne. The pathogen was recovered from pathogen-free seeds mixed with infested debris suggesting infested seed may contribute to recently documented dissemination of this pathogen worldwide. Isolates of Fusarium oxsyporum f. sp. lactucae were analyzed for genetic diversity using inter-simple sequence repeat molecular markers. Results revealed 2 main groups within the Arizona isolates corresponding to eight haplotypes in 2005, which evolved from 2 haplotypes in 2001. Haplotype 1-05 was widespread, occurring in two of the four countries where F. o. f. sp. lactucae has been reported. 23 haplotypes were identified among the California isolates that clustered into two subgroups. The clustering of isolates from Arizona suggests that there has been more than one introduction of the pathogen into Arizona.

Individual isolates of the fungus Nectria haematococca exist in a wide range of habitats and part of this diversity is attributed to the presence of conditionally dispensable (CD) chromosomes that carry habitat-defining genes. In the current study a new factor located on one of these CD chromosomes was found. This trait allows pea pathogenic isolates of N. haematococca to grow in homoserine, a compound present in large amounts on pea root exudates. The gene(s) for homoserine utilization (HUT) are located on the same CD chromosome that carries the cluster of genes for pea pathogenicity, the PEP cluster. The PDA1 gene, a member of the PEP cluster, is routinely used as a marker for the presence of this CD chromosome, therefore it has been called the PDA1-CD chromosome. For the purpose of identifying the HUT gene(s), a physical map of the PDA1-CD chromosome was constructed. This map, in combination with synteny analysis, and Southern hybridizations led to the identification of a region of 365Kb that is likely to contain the HUT gene. By searching the publicly available genome of N. haematococca several candidates for HUT were identified.The synteny evaluation between the PDA1-CD chromosome and a different CD chromosome that carries the MAK1 gene, for chickpea pathogenicity, revealed a region (&gt; 463Kb) of synteny, which advocates for a common ancestor for these CD chromosomes. However a large region (~ 1 Mb) in each of the CD chromosomes was found to carry unique DNA, therefore we proposed that individual isolates of this fungus contain large regions of unique DNA located on the CD chromosomes. The localization of syntenic regions also suggests that breakage points previous identified in the MAK1-CD chromosome could potentially be "hot spots" for recombination between both CD chromosomes. Furthermore, the anchoring of the PDA1-CD map to the genome of N. haematococca allowed the identification of additional putative habitat colonization genes present on both CD chromosomes, and niche-defining genes on the PDA1-CD chromosome.

Preeclampsia is the leading cause of foetal and maternal mortality and morbidity in developing countries. In South Africa, maize is a dietary staple for most black African populations and is susceptible to contamination by mycotoxins such as fumonisin B1 (FB1).Fumonisin B1 is a ubiquitous secondary metabolite of Fusarium fungi produced predominantly by Fusarium verticillioides. This mycotoxin shares structural similarities with the backbone of sphingoid bases (sphinganine and sphingosine) which are substrates for the biosynthesis of complex sphingolipids. The mechanism of FB1 toxicity therefore is centred on the disruption of this process. The aim of the present study was to elucidate the possible causal link between FB1 and preeclampsia. Following ethical approval, 20 normotensive and 20 preeclamptic patients were recruited into the study. Blood and placental tissue were collected and processed for further analysis. The presence of FB1 was verified using standard immunohistochemical and electrophoretic techniques. The levels of FB1 and sphingolipids were quantified using high performance liquid chromatography (HPLC). Western blotting was conducted to confirm the presence of FB1 in the serum. Placental tissue apoptosis was evaluated using Hoechst staining and other markers. Lipid peroxidation was measured in serum and placental tissue of both groups. Fumonisin B1 was immunolocalised within the endothelial cells and mesenchymal cells of placentas from both groups, while FB1 was present in cytotrophoblastic cells of preeclamptic patients only. In addition, FB1 concentrations were significantly higher in preeclamptic compared to normotensive serum samples. Sphinganine was significantly elevated in preeclamptic serum samples whilst there was no statistical difference in the sphingosine levels between the groups. Chromatin condensation was higher in the preeclamptic patients. Caspase 3 and Fas were present with greater intensity in preeclamptic samples. The levels of lipid peroxidation were significantly higher in both serum and placental tissue of preeclamptic patients. This study has demonstrated not only the presence of FB1 in the serum and placental tissues of pregnant women but also the potential effects of this mycotoxin in the humans. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2012.

No description available.

RESUMEN: “Susceptibilidad antifúngica y filogenia molecular de especies del género Fusarium de interés clínico” Fusarium solani, Fusarium oxysporum y Fusarium verticillioides, así como otras especies del género menos frecuentes, son importantes patógenos oportunistas para el hombre, para las cuales aún no existe un tratamiento eficaz. Además, la sensibilidad al tratamiento antifúngico varía en función de la especie, por lo que la identificación a nivel de especie de los aislados clínicos es de gran interés. Sin embargo, esta identificación resulta compleja en base a sus caracteristicas morfológicas. Las nuevas técnicas de biología molecular han permitido facilitar su identificación así como establecer criterios más adecuados para delimitar las diferentes especies del género. Por todo ello, en la presente tesis se ha pretendido: seleccionar los marcadores genéticos más adecuados para la identificación molecular de las especies del género; determinar la existencia de grupos filogenéticos diferenciados entre los aislados de cada una de las especies identificadas y evaluar la actividad in vitro de diferentes antifúngicos, frente a las especies de Fusarium de interés clínico y, averiguar si existen diferencias en cuanto a la sensibilidad antifúngica de los grupos filogenéticos obtenidos. El análisis multigénico realizado con aislados de F. solani ha confirmado que constituye un complejo de especies, que presenta una elevada resistencia frente a todos los antifúngicos ensayados. El análisis del gen del EF-1α ha confirmado la identificación de cuatro grupos filogenéticos dentro del complejo de F. oxysporum que han mostrado diferente sensibilidad antifúngica in vitro frente a la terbinafina. La región TUB del gen de la β-tubulina ha resultado ser un excelente marcador molecular para identificar el resto de especies de interés clínico que, en su mayoría, han mostrado sensibilidad frente a la terbinafina. / Summary: “Antifungal susceptibility and molecular phylogeny of the genus fusarium of clinical interest” Introduction. Fusarium solani, Fusarium oxysporum and Fusarium verticillioides, as well as other less frequent species of the genus, are important opportunists pathogenic to Man for which there is still no effective treatment. In addition, sensitivity to effective antifungal treatments varies depending on species, therefore identification of samples to species level is of primary interest. However, identification on the basis of morphological characteristics can be difficult. New techniques in molecular biology have made identification easier and allowed more adequate criteria to be established in order to delimit the species of the genus. Aims. To select better genetic markers for the molecular identification of the species of the genus, to determine the existence of phylogenetic groups that can delimit samples of each of the species identified, to evaluate the in vitro activity of different antifungals against species of Fusarium of clinical interest, and to find out if there are any differences with respect to the antifungal sensitivity of the phylogenetic groups obtained. Findings. Multigenetic analysis carried out with samples of F.solani have confirmed that it is a species complex that presents a high resistance against all the antifungals tested. The analysis of gene EF-1α has confirmed the identification of four phylogenetic groups within the F.oxysporum complex that have shown different antifungal sensitivity in vitro against terbinafine. The TUB region of the gene of β-tubulina has proven to be an excellent molecular marker for identifying the other species of clinical interest that have shown sensitivity primarily against terbinafine.

Growth chamber trials were performed to investigate optimal conditions for the small scale production of isolate M12-4A of Fusarium oxysporum on substrate materials that are locally available to subsistence farmers in West Africa. Field trials were conducted in Mali to evaluate the effectiveness of F. oxysporum for the control of Striga hermonthica and to determine the host range of F. oxysporum. F. oxysporum grew and colonized substrates over a range of temperatures (24, 28 and 32 C). Chopped sorghum straw pieces, straw fibres, and glumes supported abundant mycelial growth. Full colonization of the substrates was observed within 10 days. Production of infective propagules (microconidia and macroconidia) was optimum at 28 C. Optimum wetness of the substrates was obtained by soaking straw or glumes overnight. In field studies, the incorporation of 2.6 g of dried ground straw inoculum per sorghum seed pocket (120 cm$ sp2$), at a depth of 5 or 10 cm, resulted in a 60% reduction of emerged S. hermonthica 82 days after sowing. At harvest, biomass of Striga was also reduced by 70% and sorghum grain yield was almost doubled compared with the control. Sorghum, millet, maize, rice, fonio, cotton, cowpea, groundnut, okra and sorrel were immune to isolate M12-4A.

Fusarium oxysporum M12-4A fungus is being evaluated for the biocontrol of Striga hermonthica, a parasitic weed of African cereal crops. The production of M12-4A inoculum was assessed in four Malian villages using local technology and substrates. A delivery system using arabic gum to temporarily glue inoculum powder onto the crop seed was tested. In controlled conditions, coating of sorghum seeds with arabic gum and inoculum powder did not affect seed germination or inoculum viability. However, one week at 40°C significantly decreased the viability of the inoculum by 31%. Fungus growth and chlamydospore germination were also reduced by temperatures of 34 and 36°C. M12-4A was susceptible to the fungicide thiram (ED50 = 38.5mug). Field trials were conducted in Mali to evaluate the large-scale efficacy of the seed coating technology. F. oxysporum M12-4A was detected from some S. hermonthica tissue and soil samples using specific primers and Real Time PCR.