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Bananas are an extremely important crop, serving as both a staple food in developing countries and as a dessert fruit in Western society. Two of the most devastating pathogens currently affecting both commercial and subsistence banana production are Fusarium oxysporum (Foc; causal agent of Fusarium wilt) and Mycosphaerella species (causal agent of black and yellow Sigatoka). Conventional breeding programs designed to improve the disease resistance characteristics of the commercially elite Cavendish cultivar have, thus far, been largely unsuccessful. Genetic engineering is now regarded as the most promising method to generate enhanced disease resistance in banana. In other crops and model species, strategies to enhance disease resistance have included the transgenic expression of defense-related genes such as; disease resistance genes (R genes), downstream signaling genes (eg. NPR1, non-pathogenesis related) and antimicrobial peptides (AMPs). The overall aims of this research were to amplify and compare the nucleotide binding site (NBS) domains of potential disease resistance genes from disease resistant and disease susceptible banana cultivars. To isolate and compare complete R gene sequences from these cultivars. To generate transgenic Lady Finger banana plants expressing the D4E1 antimicrobial peptide under the control of two different promoters and finally to assess extracts from these plants for their ability to inhibit the growth of Foc Race1. Using degenerate primers, the NBS domains of six resistance gene candidate (RGC) sequences were amplified from the disease resistant cultivar Calcutta 4 (C4) and the disease susceptible cultivar Cavendish (Cav). The RGC 1, 2, 5 and 6 sequences showed similarity to previously characterized R gene sequences isolated from monocotyledonous plant species, while RGCs 3 and 4 showed similarity to R genes which form part of the Fusarium wilt resistance locus isolated from the dicotyledonous species, Lycopersicon esculentum; as well as other monocotyledonous R genes. RGCs 1-4 and 6 were present and transcriptionally active in both C4 and Cav, whereas RGC-5 was present in Cav only and was not transcribed. The transcripts could not be detected by Northern analysis, which is consistent with previous reports that R genes are constitutively transcriptionally active at only low levels. The NBS domains of RGCs 1-6 showed less than 65% similarity (amino acid level) to one another but when each individual RGC isolated from the C4 and Cav gDNA and cDNA templates was compared the sequences showed greater than 97% similarity (amino acid level). Comparative sequence analysis revealed amino acid positions that were consistently different between the C4 and Cav clones. Southern analysis revealed that RGC 1-5 were present in both the C4 and Cav genomes in only low copy number (1-2 gene copies with 1-3 alleles), whereas RGC-6 showed high copy number in both cultivars. Complete RGC sequences were subsequently amplified by RNA-ligase-mediated (RLM) -RACE and 3'-RACE using specific primers designed to each of the RGC 1-4 NBS domains. Amplicons for each RGC were assembled to form potentially complete RGC sequences. Analysis of the sequences revealed the presence of coiled coil (CC) motifs in two of the amino terminal sequences while leucine rich repeats (LRRs) were identified at the carboxy terminal of all sequences. Multiple 3'-RACE products were amplified for each RGC sequence. Although the polyadenylated products were of different lengths, the sequences were greater than 98% identical at the amino acid level (except an RGC 3 clone which was 91-95% identical to the other RGC 3 clones due to a 37 amino acid deletion). Specific primers used to amplify each complete RGC sequence from both C4 and Cav DNA revealed that: RGC 1 (3.53 kbp) could be amplified from both C4 and Cav; RGCs 2 (2.99 kbp) and 4 (4.44 kbp) could be amplified from only Cav, however, the proposed truncations of these sequences (RGC 2: 1.3 kbp, RGC 4: 2.8 kbp and 2.9 kbp) could be amplified from both cultivars; RGC 3 (4.57 kbp) could not be amplified from either C4 or Cav, however, the three shorter sequences (1.96 kbp, 1.34 kbp and 1.28 kbp) could be amplified from both templates. The functional significance of the truncated sequences is currently unknown, however, truncated sequences have been detected in a number of R gene families isolated from other crops. No major sequence differences, such as deletions/insertions or early stop codons, were identified between the RGC sequences amplified from C4 as compared to Cav (greater than 91% amino acid similarity) and no sequence was identified as being present in the susceptible but absent from the resistant cultivar. However, comparative analysis of multiple clones isolated from C4 and Cav did reveal amino acid residues that were consistently different between the two cultivars. These differences may result in differing resistance capabilities, functional genomics studies would need to be undertaken to determine this. It has been proposed that CC-NBS-LRR type R genes employ NDR1/HIN1-like (NHL) proteins, after pathogen invasion is detected, in the signaling process that ultimately leads to the elaboration of a defense response. A NHL partial sequence (420 bp) was amplified from the C4 banana cultivar. The complete sequence of this gene (termed NHL-1) was isolated using RLM and 3'-RACE technologies (576 bp and 535 bp amplicons, respectively) and subsequently the 1.106 kbp sequence was PCR amplified from both the C4 and Cav cultivars. The banana NHL-1 gene contained conserved motifs/domains previously identified within other NHL-type gene sequences. These included a signal peptide motif, a transmembrane domain and three previously identified conserved motifs. Based on current research into NHL type genes, the banana NHL-1 sequence may not be useful as a transgene to enhance disease resistance in elite cultivars. However, it potentially plays an important role in the defense response signal transduction pathway and therefore will further our understanding of plant-pathogen interactions in banana. Transgenic Lady Finger banana plants expressing the D4E1 antimicrobial peptide under the control of either the maize polyubiquitin (Ubi) or banana bunchy top virus (BBTV) DNA-6 (Bt6.1) promoters were generated. These plants were subsequently assessed for the ability of their crude protein extracts to inhibit the germination of Fusarium oxysporum f.sp. cubense Race1 conidia in vitro. These anti-fungal bioassays revealed that fungal colony growth was reduced by 37-100% using extracts from the pUbi-D4E1 transgenic lines and 89-99% using extracts from the pBt6.1-D4E1 transgenic lines. The transgenic lines are currently undergoing multiplication in preparation for glasshouse and small plant challenge trials for resistance to Fusarium wilt. These preliminary results suggest that D4E1 may be useful in enhancing disease resistance in banana.

Thesis (M.Sc. (Agriculture Agronomy)) --University of Limpopo, 2017 / Among the fungal diseases infecting tomato crop, early blight caused by Alternaria solani (Ellis & G. Martin) is one of the most destructive fungal foliar diseases. The aim of this current study was to document the prevailing pathogenic diversity of A. solani populations in Limpopo based on morphological characteristics, fungicide sensitivity, and aggressiveness of A. solani isolates. The tested isolates were collected from four different areas across different tomato production sites in the Limpopo Province and pathogen isolations were carried-out under laboratory conditions. The the morphological variation of different A. solani isolates was evaluated. The results obtained in this study show that A. solani isolates exhibit high variations in mycelial pigmentation, number of septa, beak length and colony diameter. The current study also evaluated the sensitivity of A. solani isolates obtained from different areas in the Limpopo to commonly used fungicides. All the tested fungicides (chlorothalonil, copper oxychloride and mancozeb) reduced the mycelial growth of A. solani isolates, even at lower concentrations. However copper oxychloride fungicide provides better inhibition of mycelial growth as compared to other tested fungicides. Furthermore, the aggressiveness of the different isolates was investigated using Money-maker and Rodade tomato cultivars. The results obtained in our, study revealed that isolates from different areas differed in terms of their level of aggressiveness on both cultivars. However, all the tested isolates were aggressive in their ability to cause early blight in both cultivars. There was a difference on how both cultivars responded to isolates from different areas with the Money-maker being highly susceptible to all the tested isolates as than the Rodade Key words: Aggressiveness, Alternaria solani, Fungicide sensitivity, isolates, Limpopo Province, Morphological characterization, susceptible tomato cultivars. / NRF (National Research Foundation)

Infectious fungal diseases pose a substantial threat to susceptible plant species, causing host declines, limiting host populations’ community role, and threatening the sustainability of natural ecosystems. Evaluating disease severity and progression is essential for understanding the impacts of these diseases, and this information could provide insights into developing future interventions. On Roan Mountain, Tennessee, native monocot Veratrum viride (Röhl.), was recently discovered to be infected with a fungal pathogen, Pseudocercosporella sublineolata (Thüm. U. Braun.), a Veratrum-specific leaf spot disease. To understand the prevalence and impacts of this disease, a demographic and disease severity study was performed. We hypothesized that P. sublineolata infection was associated with the decline of leaves and the premature seasonal senescence of V. viride plants, and that the increase in the number of leaf spots over the growing season was associated with the decline in plant health. To test these hypotheses, twenty plants from two different populations on Roan Mountain were systematically selected and tagged for a total of forty plants. From June through September 2021, demographic characteristics (plant height, number of leaves, and whether the plant flowered), and disease data (plant health, number of leaf spots, diameter of spots, and whether spots harbored P. sublineolata spores) were recorded over seven visits. To diagnose the disease, leaf spot samples were collected, slides were prepared and examined for spores at 100/200X magnification. We confirmed the diagnosis of P. sublineolata infection based on the conidia’s shape, size, and number of cells. The relationship between P. sublineolata and plant senescence was strengthened because the samples had abundant conidia that were present as pure isolates rather than a mixture of other potential microbial pathogens. All plants in the study declined progressively throughout the growing season, and they senesced earlier than expected, i.e., before the first frost. This early season decline likely limits reserves stored in the overwintering bulb and inhibits seed maturation, thereby posing a threat to the viability of these V. viride populations on Roan Mountain.