Fusarium wilt of banana has a long and devastating history in many of the world’s banana producing countries. The most pronounced damage caused by Fusarium oxysporum f.sp. cubense (Foc), the Fusarium wilt pathogen, occurred during the 20th century in Central America, where tens of thousands of virgin forests were lost to further banana production. No control strategy is effective against Fusarium wilt other than replacement of susceptible by resistant varieties. It is, therefore, important to develop or identify resistant replacements that would not only be able to resist the pathogen, but also be acceptable to consumers. Resistance in wild banana varieties has been identified, and hybrids have been developed by breeding programmes with good resistance to Fusarium wilt. These varieties, unfortunately, appear not to be acceptable replacements for Cavendish bananas, the sweet desert banana variety that serves as the primary export banana and constitutes almost 40% of all bananas planted in the world today. A field selection, GCTCV-218, now proved to be the Cavendish plant with the most resistance to Foc ‘tropical’ race 4 (VCG 0121) has saved the Cavendish-based banana industry in Taiwan from devastation. In this thesis, GCTCV-218 has been evaluated against Foc ‘subtropical’ race 4 (VCG 0120), the primary variant of the pathogen in subtropical banana-producing countries such as South Africa, Australia and the Canary Islands. Defence-associated genes that are differentially expressed and that were up-regulated early in the defence response against the pathogen were isolated and identified. Greenhouse and field trials conducted at the research facilities of the Forestry and Agricultural Biotechnology Institute, University of Pretoria and in Kiepersol, South Africa, respectively, showed that GCTCV-218 had a significantly higher level of disease tolerance against Foc ‘subtropical’ race 4 (VCG 0120) when compared to the commercially grown Williams cultivar. Phenolic assays revealed that total phenolics and cell-wall bound phenolics were expressed at higher levels in GCTCV-218 after pathogen attack and seemed to play an important role in the tolerance of GCTCV-218. It was, therefore, proposed that GCTCV-218 could be considered a replacement for other Cavendish banana varieties planted in South Africa. The genetic basis of defence mechanisms in banana to Foc is unknown. In this investigation, Suppression Subtractive Hybridisation (SSH) was used to construct a cDNA library, containing banana genes that were up-regulated early (3&6 hours after infection), in the GCTCV-218/Foc interaction. The efficiency of the procedure was confirmed by PCR amplification of a known defence gene (endochitinase) present in the subtracted tester material, as well as analysing the reduction of a known housekeeping gene, actin, in the subtracted material compared to unsubtracted material. Southern blot data further provided confidence in the subtraction process. A cDNA library containing 736 gene fragments was constructed and then subjected to a screening procedure to remove false positives that escaped the subtraction process. The screening of a banana cDNA library for defence-related genes involved the development of a high-throughput cDNA microarray technique. This novel technique removed all false positives, such as housekeeping genes that escaped the subtraction as well as clones representing rDNAs. Seventy-nine genes differentially expressed in GCTCV-218 and not in Williams were selected, sequenced and subjected to BLASTX, BLASTN and DBest searches. Of these, several gene fragments showed homology to defence-associated genes, and 20 unique genes fragments were identified. These include two different peroxidases, response regulator 6, catalase 2, metallothionein, pectin acetylesterase (PAE), two different unknown proteins, salt stress, trypsin inhibitor, unspecific monooxygenase cytochrome P450, Bowman Birk proteinase inhibitor, root control, xylanase inhibitor, inhibitor CII, hypothetical protein, putative senescence-associated protein, pathogenesis-related protein 1 (PR1) and ribosomal protein S3a. The significance of the defence reaction to Fusarium wilt diseases in agricultural crops depends on the tempo of plant response. When a host plant is able to respond early to pathogen invasion the pathogen is successfully contained, preventing further spread throughout the plant. The expression of genes with antimicrobial activity, such as endochitinase, suggests an induced biochemical defence response against Foc. The expression of PAE and PR1 results in the deposition of lignin and callose production for cell wall strengthening. Four defence associated genes (catalase 2, pectin acetyl esterase (PAE), PR-1 and endochitinase) were selected for expression profile analysis using Real-time reverse transcriptase PCR, with TaqMan® and Light Cycler technology. All four genes were shown to be differentially expressed in GCTCV-218 at 3 and 6 hrs after infection, confirming SSH results. PR-1 and PAE were induced very early (3 hrs after infection) in the GCTCV-218, while PR3 and catalase 2 followed with a significant induction at 6 hrs after infection. This study concludes that GCTCV-218 is able to respond rapidly in response to Foc infection by activating both a biochemical and structural defence mechanism. / Thesis (PhD (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive plant diseases in recorded history. The disease was first discovered in Australia in 1874 but became renowned for the severe losses it caused to export banana plantations during the 1960s in Central America. The banana export industry was saved only by replacing Gros Michel bananas, the dessert banana grown for the export market, with highly resistant Cavendish banana cultivars. Despite this apparent solution, the fungus was found to attack Cavendish bananas in the sub-tropics, where plants were believed to be predisposed to the disease by the cool winter climate. Good management practices and conventional disease management strategies have not been sufficient to reduce losses and stop the disease from spreading, and today Fusarium wilt can be found in almost all banana-producing countries of the world. Since 1988, Foc has been responsible for significant losses of Cavendish bananas in tropical Asia. The only sustainable control measure, the use of resistant varieties, is not always popular as people prefer to eat locally adopted varieties that, unfortunately, are susceptible to Foc. Sustainable Fusarium wilt management in banana depends on the improvement of existing banana cultivars or the development of novel disease management strategies. Molecular biology and biotechnology provide opportunities to introduce foreign resistance genes into existing cultivars and to develop new, environmentally friendly products that can protect susceptible bananas from Foc. Better knowledge of the Fusarium wilt pathogen, its diversity, and its mechanisms of pathogenesis will contribute significantly to developing these novel approaches for control of the disease. Molecular information on the pathogenicity of Foc, however, is limited, whereas other formae speciales of F. oxysporum have been better studied. In this thesis, Agrobacterium tumefaciens-mediated transformation of (ATMT) was employed to investigate genes responsible for pathogenicity of Foc to banana. Chapter 1 provides an overview of pathogenicity in F. oxysporum. Pathogenic and non-pathogenic forms of the fungus are first introduced to the reader, and then the biology, epidemiology and etiology of pathogenic forms of F. oxysporum are discussed. The genetic make-up and ability of the Fusarium wilt fungus to cause disease in plants concludes the first part of the review. In recent years, there has been a noted increase in the number of techniques available to study hostpathogen interactions. The second part of the review concentrates on these techniques and their applications in studying pathogenicity of the Fusarium wilt pathogen. In Chapter 2, an ATMT and screening system for Foc was developed. Five A. tumefaciens strains were evaluated for their efficiency to transform Foc with a randomly integrating vector that confers hygromycin B resistance and expression of green fluorescent protein (GFP). A small insertion mutant library of Foc was created, and a subset of transformants was characterized by determining the number of T-DNA inserts present, the location and identity of predicted genes disrupted by T-DNA insertion, and whether transformants of Foc were altered in their virulence against susceptible banana plants. In Chapter 3, the role of a known pathogenicity gene, Frp1, of the tomato pathogen F. oxysporum f. sp. lycopersici (Fol) was investigated in Foc. The first objective was to isolate and characterize the Frp1 gene in Foc, and to compare it to the homologous gene in Fol. A vector containing a modified Fol Frp1 gene was then obtained and used for targeted disruption of the gene in Foc via ATMT. Mutants in which the Frp1 gene was disrupted were then analyzed for GFP expression, culture morphology, and alterations in pathogenicity to banana. / Dissertation (MSc)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Allelopathy occurs when one organism releases a compound into the environment that affects the functioning of another organism. Scientists have long suspected that alleopathic plant compounds could offer novel, softer chemistries to the ongoing battle of controlling pests in agricultural fields. Strawberry growers rely on toxic fumigants to kill soilborne fungal pests, weeds, nematodes, and insects. Increased regulations have reduced the use of fumigants (including methyl bromide), and strawberry growers need new sustainable pest control solutions. We selected four putative allelochemicals with known fungicidal and herbicidal activity (ferulic acid, gallic acid, juglone, and p-Coumaric acid). We assessed the pesticidal activity of these plant compounds both in agar and in soil on two emerging soilborne fungal pathogens (Macrophomina phaseolina and Fusarium oxysporum f.sp. fragariae), and four annual weeds commonly found in strawberry production fields (Malva parviflora, Melilotus officinalis, Poa annua, and Senecio vulgaris). We also assayed lettuce (Lactuca sativa ‘Inferno’), which served as a positive control plant species due to its sensitivity to phytotoxic compounds. Fitted sigmoidal dose-response curves predicted EC50 and EC75 values for each combination of plant compound and pest. All plant compounds inhibited the in vitro radial mycelial growth of the two soilborne fungal pathogens in a dose-dependent manner. Fusarium oxysporum f.sp. fragariae was more sensitive to the plant compounds than Macrophomina phaseolina. Average EC50 values for the radial mycelial growth of two F. oxysporum f.sp. fragariae isolates were 75.1 parts per million by weight (ppmw) juglone, 469 ppmw p-Coumaric acid, and 687 ppmw ferulic acid. Average EC50 values for the radial mycelial growth of two M. phaseolina isolates were 196 ppmw juglone, 2869 ppmw p-Coumaric acid, and 5716 ppmw ferulic acid. The three compounds we assayed in vitro also reduced M. phaseolina colony forming unit counts in soil and the EC50 values were 476 ppmw ferulic acid, 612 ppmw juglone, and 827 ppmw p-Coumaric acid. Metconazole, the conventional fungicide control, did not inhibit M. phaseolina colony forming unit counts in soil at its label high rate. The plant compounds required similar or lower rates to inhibit colony forming units that grew from M. phaseolina overwintering structures (microsclerotia) in soil as to inhibit radial mycelial growth in vitro. Based on the EC50 value in soil assays, ferulic acid was the least expensive plant compound to apply on a per acre basis to inhibit M. phaseolina ($74,226). In F.oxysporum f.sp. fragariae soil assays, the compounds induced hormesis at lower rates and may be germination stimulant candidates. Metconazole and the high rates of every compound effectively or completely inhibited F. oxysporum f.sp. fragariae colony forming units in soil. The plant compounds were more herbicidal than fungicidal in vitro. When combining the in vitro seedling length results for L. sativa, M. parviflora, P. annua, and S. vulgaris the EC50 values differed significantly (p < .0001) and were: 47 ppmw juglone, 120 ppmw p-Coumaric acid, 189 ppmw ferulic acid, and 297 ppmw gallic acid. At least one rate of ferulic acid, juglone, and p-Coumaric acid inhibited the germination of all plant species, while gallic acid only inhibited the germination of P. annua at 1000 ppmw (p < .05). In soil, visible microbial contamination in individual wells of 24-well plates and seed dormancy made it difficult to fit curves to weed seedling length data. The soil assay L. sativa seedling length EC50 values 11 days after initial treatment were slightly higher than in vitro, although plant compounds were in the same order of phytotoxicity: 129 ppmw juglone, 616 ppmw p-Coumaric acid, 644 ppmw ferulic acid, and 1584 ppmw gallic acid. Based on the EC50 value in soil assays, the least expensive compound to inhibit L. sativa seedling length on a per acre basis was gallic acid ($21,676). Germination 26 days after initial soil treatment generally declined in a dose-dependent manner for each compound. There was a direct relationship between plant compound rate and seedling damage in soil with the higher rates of all compounds, except p-Coumaric acid, inducing damage comparable to a conventional herbicide (pendimethalin or oxyfluorfen). Contaminated treatments appeared to be due to an interaction between plant compounds and microorganisms because herbicide and water controls had almost no microbial growth 11 days after initial treatment. Further, there was a significant positive linear relationship between level of contamination in phenolic acid-treated wells (ferulic acid, gallic acid, and p-Coumaric acid, p < .0001) and the in soil rate. This relationship was slightly negative in juglone soil treatments (p = .0167), which may be due to its greater antimicrobial activity than the phenolic acids. We propose that herbicidal effects in soil were due to the joint effect of the plant compounds themselves, and the microbial growth in wells. Microbial growth was either antagonistic or additive to the inhibitory action of the plant compounds. The plant compounds we assayed were inhibitory of emerging fungal pathogens in strawberry production and common annual strawberry field weeds. Evidence presented in this thesis correlates well with past research that not only found plant compounds to be herbicidal and fungicidal, but also described their modes-of-action (such as the production of reactive oxygen species that causes necrotic lesions on roots, and inhibition of glycolytic enzyme activity that prevents germination), and implicate plant compounds as carbon sources for a variety of microorganisms. Compound prices are currently exorbitant, but may decline as demand increases. Whether or not they provide effective pest control may depend on soil texture, organic matter, microbial diversity, and other edaphic factors.

No description available.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-01-10T09:15:32Z No. of bitstreams: 2 Dissertação - Nayane Oliveira Almeida - 2016.pdf: 4246837 bytes, checksum: 2ee0e2f0ece0d114ea4032c91be4556f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-10T10:58:54Z (GMT) No. of bitstreams: 2 Dissertação - Nayane Oliveira Almeida - 2016.pdf: 4246837 bytes, checksum: 2ee0e2f0ece0d114ea4032c91be4556f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-10T10:58:54Z (GMT). No. of bitstreams: 2 Dissertação - Nayane Oliveira Almeida - 2016.pdf: 4246837 bytes, checksum: 2ee0e2f0ece0d114ea4032c91be4556f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The problems caused by nematodes and by the Panama disease on banana plantations are responsible for production losses and limiting to its cultivation. In the state of Goiás there is few information about the nematode genus that affect this crop, and its relationship with the incidence of the fungus Fusarium oxysporum f. sp. cubense (Foc). This research aimed to survey the occurrence of plant parasitic nematodes, the incidence of Foc and soil attributes, and determine if there is a correlation among these factors. In January 2015, twelve banana producing regions in the state of Goiás were sampled: Anápolis, Caiapônia, Goiatuba, Itaguaru, Itumbiara (two areas), Jataí, Morrinhos, Ouro Verde, Palestina, Taquaral and Uruana. All sampled areas, except Morrinhos, revealed contamination with Foc, and all had different genus of nematodes. Meloidogyne sp., Helicotylenchus sp. and Rotylenchus sp. were the main genus of plant parasitic nematodes present in the banana plantations, with Meloidogyne sp. and Rotylenchus sp. the most dominant and abundant genus. We found that Pratylenchus sp. increases the population levels of F. oxysporum and that Helicotylenchus sp. has been affected by the concentration of P, Ca, Mn and the soil pH. / Os problemas fitossanitários causados por nematoides e pela doença mal-do-Panamá, na cultura da banana, são responsáveis por grandes perdas de produção ou são fatores limitantes de seu cultivo. Em Goiás, são escassas as informações sobre os gêneros de nematoides que afetam a bananicultura, bem como sua relação com a incidência do fungo Fusarium oxysporum f. sp. cubense (Foc), agente causal do mal-do-Panamá. Assim, esse trabalho teve por objetivo fazer levantamento da ocorrência de fitonematoides, da incidência de Foc e dos atributos dos solos, e determinar se há correlação entre estes fatores. Em janeiro de 2015, foram amostradas doze regiões produtoras de banana no estado de Goiás, distribuídas em onze municípios: Anápolis, Caiapônia, Goiatuba, Itaguaru, Itumbiara, Jataí, Morrinhos, Ouro Verde, Palestina, Taquaral e Uruana. Todas as áreas amostradas, exceto a do município de Morrinhos, apresentaram-se contaminadas com Foc, e todas apresentaram diversos gêneros de fitonematoides. Meloidogyne sp., Helicotylenchus sp. e Rotylenchus sp. foram os principais gêneros de fitonematoides presentes nos bananais no estado de Goiás, sendo Meloidogyne sp. e Rotylenchus sp. os gêneros mais dominantes e abundantes. Foi constatado que a presença de Pratylenchus sp. aumenta o nível populacional de F. oxysporum e que Helicotylenchus sp. é afetado pelos teores de P, Ca, Mn e pelo pH do solo.

Made available in DSpace on 2016-12-08T16:44:49Z (GMT). No. of bitstreams: 1 PGPV14MA158.pdf: 622907 bytes, checksum: 4bef911008ae5cffa26706180f710db4 (MD5) Previous issue date: 2014-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The addition and incorporation of organic matter to the soil, besides favoring crops by improving soil physical, can increase nutrients and add specific biochemicals capable of renewing the native microflora and microfauna. These compounds may, depending on the organic material to act as a suppressant effect and biocontrol. The aim of this study was to evaluate the effect of swine manure compost (DSC) in an orchard of apple, pear and grape vines on the population dynamics of Verticillium dahliae, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides and Trichoderma sp. Soil samples for quantification of fungal colonies and nutrient analysis were collected at a depth of 0-10 cm soil of the orchard with apple, pear and grape vines. The population of pathogenic soil fungi and Trichoderma sp. were obtained by dilution and plating of 10 g of soil samples from soil orchard who received two doses of DSC (50 to 100%) compost and two (50 and 100%), using two culture media (BDA potato-dextrose-agar) and Sabouraud-ágar-chloramphenicol. The application of different doses of DSC and chemical fertilizer began in December 2012, repeated at intervals of 60 days until the 2014 harvest analysis of macronutrients (nitrogen, phosphorus, potassium, calcium and magnesium) and micronutrients (iron, copper, zinc and Manganese) DSC and chemical fertilizer were run using Mehlich-1, spectrophotometry, acid-base titration and Kjeldahl method, all described by Tedesco et al. (1995). Results in the concentration of nutrients was related to the population of Verticillium dahliae, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides and Trichoderma sp. The experimental design was completely randomized, factorial 2 x 5, repeated in time (months). The data were analyzed using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, v.9.2) and mean comparisons using Tukey least significant difference p &#8804; 0.05. In the apple orchard, Fusarium oxysporum and Fusarium solani showed higher populations in Q100 treatments (0-110 x 103 CFU / g of soil) and Q50 (0-70 x 103 CFU/g of soil) respectively. There were differences in the population periods. Phosphorus, Potassium and Sodium showed significant differences among the treatments tested. In the orchard of pear trees the largest population of Fusarium solani was the S100 treatment (0-50 x 103 CFU/ g of soil). Treatments Q50 and Q100 had higher populations of Verticillium dahliae, Fusarium oxysporum and Fusarium verticillioides in different periods. Concentrations of Nitrogen and Potassium differ between treatments tested. In vineyards the largest populations of Fusarium solani and Fusarium oxysporum were found in December-2012 periods (0-70 x 103 CFU / g of soil) and August 2013 (0-60 x 103 CFU / g of soil) respectively. Concentrations of potassium, phosphorus and sodium were higher in treatment S50 and S100. The orchard of apple, pear and grape vines have different response to chemical and organic fertilization. The intensity of response to fertilization has little influence population dynamics of plant pathogens in soil and Trichoderma / A adição e incorporação de matéria orgânica ao solo, além de favorecer as culturas pela melhoria física do solo, podem potencializar nutrientes e adicionar compostos bioquímicos específicos capazes de renovar a microfauna e microflora nativas. Estes compostos podem, dependendo do material orgânico, agir como efeito supressor e como biocontrole. O objetivo deste trabalho foi avaliar o efeito da aplicação de dejeto suíno compostado (DSC) em pomar de macieiras, pereiras e videiras, sobre a dinâmica populacional de Verticillium dahliae, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides e Trichoderma sp. Amostras de solo para quantificação de colônias fúngicas e análise de nutrientes foram retiradas na profundidade 0-10 cm de solo do pomar de macieiras, pereiras e videiras. A população de fungos fitopatogênicos de solo e Trichoderma sp. foram obtidas pela diluição e plaqueamento de 10 g de amostras de solo provenientes do solo do pomar que receberam duas doses de DSC (50 e 100%) e duas de adubo químico (50 e 100%), utilizando dois meios de cultura, BDA (batata-dextrose-agar) e Sabouraud ágar-cloranfenicol. A aplicação das diferentes doses de DSC e adubo químico tiveram início em dezembro-2012, repetidas em intervalos de 60 dias até a safra 2014. A análise dos macronutrientes (nitrogênio, fósforo, potássio, cálcio e magnésio) e micronutrientes (ferro,cobre,zinco e Manganês) do DSC e da adubação química foram realizados pelos métodos de Mehlich -1, espectrofotometria, titulação ácido-base e método Kjeldahl, todas descritas por Tedesco et al. (1995). Resultados da concentração de nutrientes foi relacionado à população de Verticillium dahliae, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides e Trichoderma sp. O delineamento experimental foi inteiramente casualisado, em arranjo fatorial 2 x 5, repetidos no tempo (meses). Os dados foram analisados pelo procedimento MIXED do SAS (SAS Inst. Inc., Cary, NC, v.9.2) e as comparações de médias usando a diferença mínima significativa de Tukey p &#8804; 0,05. No pomar de macieiras, Fusarium oxysporum e Fusarium solani apresentaram maiores populações nos tratamentos Q100 (0-110 x 103 UFC/g de solo) e Q50 (0-70 x 103 UFC/g de solo) respectivamente. Houve diferenças da população nos períodos avaliados. Fósforo, Potássio e Sódio apresentaram diferenças significativas entre os tratamentos testados. No pomar de pereiras a maior população de Fusarium solani foi ao tratamento S100 (0-50 x 103 UFC/g de solo). Os tratamentos Q50 e Q100 apresentaram maiores populações de Verticillium dahliae, Fusarium oxysporum e Fusarium verticillioides em diferentes períodos de avaliação. Concentrações de Potássio e Nitrogênio apresentaram diferenças nos tratamentos testados. Na cultura da videira as maiores populações de Fusarium solani e Fusarium oxysporum foram encontradas nos períodos dezembro-2012 (0-70 x 103 UFC/g de solo) e agosto-2013 (0-60 x 103 UFC/g de solo) respectivamente. Concentrações de Potássio, Fósforo e Sódio foram superiores nos tratamento S50 e S100. O pomar de macieiras, pereiras e videiras apresentam diferentes resposta a adubação química e orgânica. A intensidade de resposta da adubação pouco influencia a flutuação da população de fitopatógenos de solo e Trichoderma

Submitted by Onia Arantes Albuquerque (onia.ufg@gmail.com) on 2018-10-08T13:49:50Z No. of bitstreams: 2 Tese - Stella Cristina Dias Valdo - 2017.pdf: 4757114 bytes, checksum: 62d9148c9c6315952ac5a86bdb1f9417 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Rejected by Luciana Ferreira (lucgeral@gmail.com), reason: Olhe o espaço a mais na citação: VALDO, S. C. D. Estudo de resistência à murcha-de-fusarium e identificação de QTLs em feijeiro-comum. 2017. 178 f. Tese (ESPAÇO A MAIS Doutorado em Genética e Melhoramento de Plantas) - Universidade Federal de Goiás, Goiânia, 2017. on 2018-10-09T10:59:43Z (GMT) / Submitted by Onia Arantes Albuquerque (onia.ufg@gmail.com) on 2018-10-09T11:17:13Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese - Stella Cristina Dias Valdo - 2017.pdf: 4757114 bytes, checksum: 62d9148c9c6315952ac5a86bdb1f9417 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-10-09T11:41:12Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese - Stella Cristina Dias Valdo - 2017.pdf: 4757114 bytes, checksum: 62d9148c9c6315952ac5a86bdb1f9417 (MD5) / Made available in DSpace on 2018-10-09T11:41:12Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese - Stella Cristina Dias Valdo - 2017.pdf: 4757114 bytes, checksum: 62d9148c9c6315952ac5a86bdb1f9417 (MD5) Previous issue date: 2017-04-27 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The common bean (Phaseolus vulgaris) crop plays an important role in the culture and economy of Brazil. It is cultivated in all Brazilian regions and is affected by several diseases like fusarium wilt which is caused by Fusarium oxysporum f. sp. phaseoli (soil-born fungus). This disease brings significant losses in common bean culture and genetic resistance is the primary form of control. One of the core goals of breeding programs is the development of resistant cultivars, therefore the objectives of this work are: i) To select F. oxysporum f. sp. phaseoli resistant F5:7 lines resulted from the crossing between Ouro Branco X CNFP10132, under controlled field and environment conditions ii) To identify SSR markers and QTL-linked SNPs associated with the resistance of common bean to fusarium wilt using 92 recombinat inbred lines(RILs) resulted from the crossing between Ouro Branco x CNFP10132. In the first study, 140 lines, the breeders Ouro Branco and CNFP10132, BRS Esplendor (resistant) and BRS Supremo (susceptible) as controls were evaluated. Field trials were conducted in a center pivot area where natural infestation of the pathogen occurs. The treatments were evaluated in summer and winter crop and the experimental design used was 12x12 triple lattice. The two controlled environment trials were conducted in a completely randomized design. The treatments were inoculated by cutting and immersing the roots in a conidial suspension, which was adjusted to 1x106 conidia/ml for five minutes. The evaluation was performed using a scale of nine grades that represent the severity of the disease: 1 – absence of symptoms and 9 – over 75% of foliage with wilt symptoms. Data were submitted to analysis of variance and Scott-Knott test for both environments. The area under the disease progress curve (AUDPC) and genetic parameters were estimated for controlled environment tests. Significant differences were observed for crops and for controlled environment trials, indicating that environment influences directly the severity of the disease. Highly significant differences were found for lines in all environments evaluated, demonstrating the existence of genetic variability, which allows the selection of resistant lines resistant to fusarium wilt. Treatments were classified in different groups according to the Scott-knott test. When considering the lowest averages in field, controlled environment and AUDPC, the strains Ouro Branco x CNFP 10132.140, Ouro Branco x CNFP 10132.49, Ouro Branco x CNFP 10132.12, Ouro Branco x CNFP 10132.90 and Ouro Branco x CNFP 10132.48 were prominent and are candidates to produce a breeding program. Heritability estimates were high for all environments, mean of 85.48% for field and 95.47% for controlled environment. Therefore, selection for resistance to F. oxysporum f. sp. phaseoli of these lines, will be successful. In the second study it was extracted DNA from 92 lines and from genitors for genotyping with SSRs and SNPs. In order to obtain the localization of these markers, sequences of the primers were aligned to the andean genome of the common bean. The method of single marker (analysis of QTLs based on linear regression) was used to identify QTLs associated with fusarium wilt resistance. These markers were considered significant when brought up p-value <0.05. Ninety-three markers were linked to 104 QTLs associated with fusarium wilt resistance and among these, were considered significant in more than one environment PV 115, PV 251, BARC-PV-0004089, BARC-PV-0004548, BARC-PV-0003450, BARC-PV-0006051, BARC-PV-0003368 , BARC-PV-0005477 and BARC-PV-0004897. However only the BARC-PV-0003450 marker was highly significant in the two environment controled trials (p <0.001) and winter crop (p <0.01) and explained up to 21.5% of the phenotypic variance. Subsequently, the gene annotation was made considering the location of all markers that were significant at p <0.01 comprising 500 kb before and after the localization. 960 coded transcripts were annotated. It was observed in gene annotation that BARC-PV-0003450 marker is located on the chromosome 8, 338.54 kb distant of the gene Phvul.008G014700 which is associated with the putative protein RPP13 related to disease resistance, identified in Arabidopsis thaliana. This protein belongs to the third class of resistance genes that encloses the domain called Leucine-Rich Repeats (LRR). This domain is involved in the recognition of the pathogen by the host during the infection process. Therefore, this marker is suitable for marker- assisted selection aiming the development of cultivars resistant to fusarium wilt. / A cultura do feijoeiro-comum (Phaseolus vulgaris) tem importância cultural e econômica no Brasil. O feijoeiro-comum é cultivado em todas as regiões brasileiras e é acometido por várias doenças, como a murcha-de-fusarium, causada pelo fungo habitante de solo Fusarium oxysporum f. sp. phaseoli. Esta doença causa significativas perdas na cultura e a principal forma de controle é a resistência genética. Desenvolver cultivares resistentes é um dos alvos dos programas de melhoramento, portanto os objetivos deste trabalho foram: i) selecionar linhagens resistentes obtidas de população F5:7 oriunda do cruzamento entre Ouro Branco e CNFP10132 para F. oxysporum f. sp. phaseoli, em condições de campo e de ambiente controlado e ii) identificar marcadores SSR e SNP's ligados a QTLs associados à resistência do feijoeiro-comum à murcha-de-fusarium utilizando 92 linhagens recombinantes endogâmicas (RILs) derivadas do cruzamento Ouro Branco x CNFP10132. No primeiro estudo 140 linhagens, os genitores Ouro Branco e CNFP10132, duas testemunhas BRS Esplendor (resistente) e BRS Supremo (suscetível) foram avaliados. Os ensaios de campo foram conduzidos em área de pivô central onde ocorre infestação natural do patógeno. Os tratamentos foram avaliados em duas safras (safra das águas e de inverno) em delineamento de látice triplo 12x12. Os dois ensaios em ambiente controlado foram conduzidos em delineamento inteiramente causalizado. As plantas foram inoculadas utilizando o método de corte de raiz e imersão destas na suspensão de conídios, que foi ajustada para 1x106 conídeos/mL durante cinco minutos. A avaliação foi feita utilizando uma escala de notas de nove graus que representam a severidade da doença: sendo 1 - ausência de sintomas e 9 - acima 75% da folhagem com sintomas de murcha. Os dados foram submetidos à análise de variância e teste de Scott-Knott para os ambos ambientes. Para os ensaios em ambiente controlado foram estimados área abaixo da curva do progresso da doença (AACPD) e parâmetros genéticos. Foram observadas diferenças significativas para safras e para ensaios de ambiente controlado, indicativo de que o ambiente influencia diretamente na severidade da doença. Foram encontradas diferenças altamente significativas para linhagens em todos os ambientes avaliados, evidenciando a existência de variabilidade genética, o que possibilita seleção de linhagens resistentes à murcha-de-fusarium. Ao considerar as menores médias em campo, ambiente controlado e ACCPD as linhagens Ouro Branco x CNFP 10132.140, Ouro Branco x CNFP 10132.49, Ouro Branco x CNFP 10132.12, Ouro Branco x CNFP 10132.90 e Ouro Branco x CNFP 10132.48 se destacaram e são candidatas para compor o programa de melhoramento. As estimativas de herdabilidade foram altas para todos os ambientes, média de 85,48% para campo e 95,47% para ambiente controlado. Portanto, a seleção para resistência à F. oxysporum f. sp. phaseoli dentre estas linhagens, será bem sucedida. No segundo estudo foi extraído o DNA de 92 linhagens e dos genitores para genotipagem com marcadores SSRs e SNPs. Para obtenção da localização destes marcadores as sequências dos primers foram alinhadas no genoma andino do feijoeiro-comum. O método de mapeamento por marcas simples (análise de QTLs por meio da regressão linear) foi utilizado para identificar QTLs associados à resistência à murcha-de-fusarium. Foram considerados marcadores significativos os que apresentaram p-valor<0,05. Noventa e três marcadores foram identificados ligados a 104 QTLs associados à resistência à murcha-de-fusarium. Dentre estes marcadores destaca-se os que foram significativos em mais de um ambiente PV 115, PV 251, BARC-PV-0004089, BARC-PV-0004548, BARC-PV-0003450, BARC-PV-0006051, BARC-PV-0003368, BARC-PV-0005477 e BARC-PV-0004897. Dentre os marcadores, somente o marcador BARC-PV-0003450 foi altamente significativo nos dois ensaios, em ambiente controlado (p<0,001) e na safra de inverno (p<0,01), e explicou até 21,5% da variância fenotípica. Foi feita a anotação gênica considerando a localização de todos os marcadores que foram significativos à p<0,01 e abrangeu 500 kb anterior e posterior à localização. Foram anotados 960 transcritos codificados. Ainda observou-se que o marcador BARC-PV-0003450 está localizado no cromossomo 8 distante 338,54 kb do gene Phvul.008G014700 o qual está associado à proteína putativa RPP13 relacionada com resistência à doenças, identificada em Arabidopsis thaliana. Esta proteína pertence à terceira classe de genes de resistência que engloba o domínio denominado de Repetições Ricas em Leucina (LRR; Leucine Rich Repeats). Este domínio está envolvido no reconhecimento do patógeno pelo hospedeiro durante o processo de infecção. Portanto há a possibilidade de selecionar linhagens resistentes à murcha-de-fusarium e identificar QTLs que possivelmente estão ligados aos marcadores utilizados

La vanille, utilisée en alimentation et parfumerie, est une orchidée originaire d’Amérique centrale et maintenant cultivée dans l’océan indien (Vanilla planifolia, 97% de la production mondiale) et dans l’océan pacifique (Vanilla tahitensis, 3% de la production mondiale, essentiellement en Polynésie française).J’ai montré que Vanilla tahitensis était aussi sensible que Vanilla planifolia à la fusariose et représentait la maladie principale de la vanille en Polynésie française comme dans le reste des zones de production des vanilles aromatiques. J’ai participé à démontrer qu’il s’agissait d’une nouvelle espèce de Fusarium oxysporum « f. sp. radicis-vanillae » car elle ne s’attaque qu’au cortex racinaire et ne pénètre que peu dans les tissus vasculaires. J’ai suivi pendant 4 ans l’évolution qualitative et quantitative des dégâts dus à 6 pathogènes et ravageurs dans 51 plantations à Raiatea, principalement des ombrières. J’ai ainsi montré que, contrairement aux autres pathologies et ravageurs, la fusariose connaissait un développement presqu’exponentiel ces 3 dernières années, comme c’est également le cas sur Vanilla planifolia dans le reste du monde. Ayant, par ailleurs, conduit un travail d’enquête auprès des producteurs, j’ai indiqué (l’analyse statistique et la modélisation afférente ne sont pas terminées) que le soin apporté aux lianes par les producteurs était essentiel au bon contrôle de la fusariose. / Vanilla species, used for aroma and flagrances, are orchidaceae originated from central America. They are now mostly cultivated in the Indian ocean (Vanilla planifolia, 97%, mainly in Madagascar, Indonesia, India and smaller islands from the Indian ocean) and in the Pacific ocean (Vanilla tahitensis, 3%, mainly French Polynesia).I have shown that Vanilla tahitensis was as susceptible as Vanilla planifolia towards fusarium root rot. It does represent the major losses on Vanilla tahitensis in Raiatea like reported for all the other areas of vanilla production worldly. I have participated to show that this fungus was delimited to root cortex maceration and did not invade vascular tissues, reason for which we use a new name for this pathogen : Fusarium oxysporum f sp radicis-vanillae.I have followed the etiological and epidemiological characteristics of this disease and of 5 other pathogens and insects on 51 Vanilla plantations, mainly shade-houses, during 4 years, for each vine in cohort analysis manner. I have shown, among this 6 biological causes of loss, fusarium root rot was the only one to present an exponential trajectory within the last 3 years, while the other causes remain stable or display a year increase because of climatic reasons. I did interview all the producers owning these plantations and analyze statistically the answers to the very numerous questions. The statistical and modelling analysis is not yet finished. Nevertheless, I can already claim that regular vine cleaning is absolutely necessary to maintain the vanilla plantations healthy, something which, unfortunately is not a rule for everyone.

: Les plantes ont développé au cours de leur évolution un système d’immunité innée constitué de barrières préformées et de réponses de défense induites contre les agents pathogènes. Ce travail s’inscrit dans l’étude des résistances induites chez Arabidopsis thaliana soit naturellement contre les agents pathogènes racinaires Fusarium oxysporum spp. (Fo), ou après application de phosphite (Phi), contre le parasite foliaire Hyaloperonospora arabidopsidis (Hpa). Dans la première partie du travail, les rôles des métabolites secondaires (MS) et des formes réactives de l’oxygène (ROS) dans les résistances racinaires basale et non-hôte, respectivement aux formes spéciales conglutinans (Foco) et melonis (Fom) de Fo, ont été analysés. Nous avons démontré la participation de la camalexine, une phytoalexine indolique, dans la résistance basale d’Arabidopsis à Foco. En revanche, la scopolétine, une phytoalexine phénolique, et les ROS jouent des rôles essentiels dans la résistance non-hôte à Fom. Ces données soulignent le rôle clé des MS et des ROS dans les résistances hôte et non-hôte d’Arabidopsis. Dans une deuxième partie de ce travail, le mode d’action du Phi, un oxyanion de l’acide phosphoreux (H3PO3) protégeant Arabidopsis contre l’isolat Noco2 de Hpa, a été étudié. L’effet de faibles doses de Phi est aboli chez des mutants d’Arabidopsis affectés dans la voie de transduction du signal acide salicylique (SA) indiquant que l’induction de résistance à Hpa est médiée par des mécanismes dépendants du SA. Le Phi potentialise les réponses de défense contre Hpa Noco2 via EDS1-PAD4, deux composants essentiels de la résistance basale, NPR1 et la protéine de défense PR1. L’expression de la MAP kinase MPK4, un régulateur négatif de la résistance à Hpa, est diminuée par le Phi, lors de l’inoculation par Hpa Noco2. Nos résultats démontrent que la potentialisation des réponses de défense par le Phi est associée à la régulation négative de MPK4. / Plants have developed during their evolution an innate immunity system consisting of preformed barriers and induced defence responses against pathogens. This work studies resistances in Arabidopsis thaliana induced either naturally against the root pathogen Fusarium oxysporum spp. (Fo), or after application of phosphite (Phi) against the leaf pathogen Hyaloperonospora arabidopsidis (Hpa). In a first part, roles of secondary metabolites (SM) and reactive oxygen species (ROS) in basal and non-host resistances of roots to the special forms conglutinans (Foco) and melonis (Fom) of Fo, respectively, were analyzed. We demonstrated the involvement of the indolic phytoalexin camalexin, in basal resistance of Arabidopsis to Foco. In contrast, the phenolic phytoalexin, scopoletin, and ROS play essential roles in non-host resistance to Fom. These data underscore the key role of MS and ROS in basal and non-host resistances of Arabidopsis. In a second part, the mode of action of Phi, an oxyanion of phosphorous acid (H3PO3) protecting Arabidopsis against the Hpa isolate Noco2 was studied. Effect of low doses of Phi is abolished in Arabidopsis mutants affected in salicylic acid (SA) signalling, indicating that induced resistance to Hpa is mediated by SA-dependent mechanisms. Phi primes defence responses against Hpa Noco2 via EDS1-PAD4, two essential components of basal resistance, as well as NPR1 and PR1. Expression of the MAP kinase MPK4, a negative regulator of resistance to Hpa, is decreased by Phi after inoculation with Hpa Noco2. Our results demonstrate that priming of defence responses by Phi is associated with down-regulation of MPK4.

Thesis (PhDAgric (Plant Pathology))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: In the Western Cape onion industry in South Africa, Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) has been identified as the leading cause of harvest and storage losses. This pathogen is of world-wide importance and causes Fusarium basal rot of onions (Allium cepa), affecting all onion growth stages. No information is available on the evolution, genetic diversity, molecular detection and inoculum sources of the South African Focep population. Similar to what is the case for South Africa, limited information is available on Focep in other regions of the world. World-wide, four vegetative compatibility groups (VCGs) and two single-member VCGs (SMVs) have been identified among two Japanese and 19 Colorado (USA) isolates. This polyphyletic origin of Focep suggested by VCG analyses was confirmed through molecular analyses of isolates from a few countries. Only the mating type (MAT)1-1 idiomorph has been reported for Focep isolates from Welsh onion (Allium fistulosum). The development of sustainable management strategies of Focep is dependent on knowledge of (i) the genetic diversity and evolution of Focep, (ii) whether high throughput molecular methods can be developed for identifying the most virulent and widespread Focep genotypes and (iii) the role of seedlings and seeds as primary inoculum sources, and the Focep genotypes associated with these growth stages. Therefore, the three main aims of the current study were to investigate the aforementioned three aspects. In the first aim of the study, the genetic diversity and evolution of Focep was investigated using a collection of 79 F. oxysporum isolates from South Africa (27 Focep and 33 non-pathogenic isolates) and Colorado (19 Focep isolates). VCG analyses revealed the presence of six VCGs, four among the Colorado Focep isolates (VCGs 0421, 0422, 0423 and 0424) and two among the South African bulb-associated isolates (VCGs 0425 and 0426). VCG 0421 and VCG 0425 were the two main VCGs in Colorado and South Africa, respectively. Four SMVs and one heterokaryon selfincompatible (HSI) isolate were also identified. The polyphyletic nature of Focep in South Africa and Colorado was shown through a combined translation elongation factor 1α (EF-1α) and mitochondrial small-subunit (mtSSU) phylogeny. The phylogeny divided the Focep isolates into two main clades, of which one contained the two main VCGs (0421 and 0425), SMVs and non-pathogenic isolates. The second, ancestral clade contained the HSI isolate, VCGs 0422, 0423 and 0424, and non-pathogenic isolates. Unlike the clade containing the two main VCGs, which were highly virulent toward onion bulbs, the ancestral clade contained isolates that were mostly moderately virulent. The incongruence of the EF-1α and mtSSU datasets with an intergenic spacer (IGS) region data set, and the presence of both MAT idiomorphs within the same isolate for some isolates, suggested possible exchange of genetic material between isolates. The second aim of the study was to develop molecular methods for identifying the two main Focep VCGs (0425 and 0421), using DNA fingerprinting methods and sequence-characterized amplified region (SCAR) markers. These techniques were first developed using the F. oxysporum isolates from the first aim, and were then used to investigate the prevalence of VCG 0425 among 88 uncharacterized F. oxysporum isolates from onion bulbs in South Africa. Two random amplified polymorphic DNA primers provided two diagnostic amplicons for VCG 0425, but attempts to develop SCAR markers from these amplicons were unsuccessful. In contrast, an interretrotransposon amplified polymorphism (IRAP) fingerprinting method enabled the developed of a multiplex IR-SCAR polymerase chain reaction method that detected the VCG 0421, 0425 and SMV 4 isolates as a group. Fingerprinting and SCAR marker testing of the 88 uncharacterized F. oxysporum isolates from South Africa (65 Focep and 23 non-pathogenic) confirmed that VCG 0425 is the main VCG in South Africa associated with mature onion bulbs, since 63 of the Focep isolates had the molecular characteristics of VCG 0425. The third aim of the study was to determine whether seed and seedling transplants are inoculum sources of Focep, and whether the same genotype (VCG 0425) that dominated on mature bulbs could be detected from these sources. Focep isolates were obtained from seven of the 13 investigated onion seed lots, as well as from onion seedling transplants that were collected from all five onion nurseries in the Western Cape. Focep seedling infection more than doubled from the 6-week growth stage to the 14-week growth stage. Seed infections by Focep were low, but the seedborne nature of Focep was confirmed by showing that a green fluorescent protein labelled Focep transformant could be transmitted from infected soil to onion seed via the onion bulbs and seedstalks. It is thus clear that commercial seed and seedlings are inoculum sources of Focep. However, the Focep genotypes on seed and seedlings are different from those in mature bulbs and were not dominated by VCG 0425. Furthermore, most (≤ 60%) of the seed and seedling isolates were moderately virulent, as compared to the mostly highly virulent isolates from mature bulbs. / AFRIKAANSE OPSOMMING: In die Wes-Kaapse uiebedryf in Suid-Afrika is Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) geïdentifiseer as die vernaamste oorsaak van oes- en opbergingsverliese. Hierdie patogeen is van wêreldwye belang; dit veroorsaak Fusarium-bolvrot van uie (Allium cepa) en affekteer alle plantgroeistadia. In Suid-Afrika is daar geen inligting beskikbaar oor die evolusie, genetiese diversiteit, molekulêre opsporing en inokulumbronne van die Focep-populasie nie. Soortgelyk aan wat die geval in Suid-Afrika is, is daar beperkte inligting beskikbaar oor Focep in ander wêrelddele. Wêreldwyd is daar vier vegetatiewe versoenbaarheidsgroepe (VVGe) en twee enkellid VVGe (ELVe) geïdentifiseer onder twee Japannese en 19 Colorado (VSA) isolate. Hierdie veelvuldige oorsprong van Focep wat deur VVG-analise voorgestel was, is deur die molekulêre analises van isolate uit ’n paar ander lande bevestig. Slegs die paringstipe (PT)1-1 idiomorf is vir Focep-isolate uit Walliese-tipe uie (ook bekend as ‘lenteuie’ in Suid Africa) (Allium fistulosum) berig. Die ontwikkeling van volhoubare bestuurstrategieë vir Focep steun op kennis van (i) die genetiese diversiteit en evolusie van Focep, (ii) of hoë-deurset molekulêre metodes ontwikkel kan word vir die identifisering van die mees virulente en wydverspreide Focep-genotipes en (iii) die rol van saailinge en saad as primêre inokulumbronne, en die Focep-genotipes wat met hierdie groeistadia geassosieer word. Daarom was die hoof doelstellings van hierdie studie om die bogenoemde drie aspekte te bestudeer. Om die eerste doel van die studie te bereik is die genetiese diversiteit en evolusie van Focep bestudeer deur gebruik te maak van ‘n versameling van 79 F. oxysporum-isolate uit Suid-Afrika (27 Focep en 33 nie-patogeniese isolate) en uit Colorado (19 Focep-isolate). VVG-analises het die teenwoordigheid van ses VVGe aangetoon – vier onder die Colorado Focep-isolate (VVGe 0421, 0422, 0423 en 0424) en twee onder die Suid-Afrikaanse bol-geassosieerde isolate (VVGe 0425 en 0426). VVG 0421 en VVG 0425 was die twee hoof VVGe in onderskeidelik Colorado en Suid-Afrika. Vier ELVe en een meerkernige self-onversoenbare (MSO) isolaat is ook geïdentifiseer. Die veelvuldige oorsprong van Focep in Suid-Afrika en Colorado is ook aangetoon deur ‘n gekombineerde translasie verlengings faktor 1α (VF-1α) en mitokondriale klein-subeenheid (mtKSE) filogenie. Dié filogenie het die Focepisolate in twee groepe verdeel, waarvan die een groep die twee hoof VVGe (0421 en 0425), ELVe en nie-patogeniese isolate bevat het. Die tweede, basal groepering het die MSO-isolaat, VVGe 0422, 0423 en 0424, en nie-patogeniese isolate bevat. In teenstelling met die eersgenoemde groepering wat hoogs virulente isolate van uiebolle bevat het, het die basale groepering isolate bevat wat meestal matig virulent was. Die inkongruensie van die VF-1α en mtKSE-datastelle met ‘n intergeen-gespasieerde (IGS) area datastel – asook die teenwoordigheid van beide PT-idiomorwe binne dieselfde isolaat by sommige isolate – het op ’n moontlike uitruiling van genetiese materiaal tussen isolate gedui. Die tweede doel van die studie was om molekulêre metodes te ontwikkel vir die identifisering van die twee hoof Focep VVGe (0425 en 0421) deur gebruik te maak van DNA-vingerafdrukke en nukleotied-gekarakteriseerde geamplifiseerde area (NKAA) merkers. Hierdie tegnieke is ontwikkel deur van die F. oxysporum-isolate van die eerste doelstelling gebruik te maak en is daarna gebruik om die frekwensie van VVG 0425 onder 88 ongekarakteriseerde F. oxysporum-isolate van uiebolle in Suid-Afrika te ondersoek. Twee gerandomiseerde geamplifiseerde polimorfiese DNS (RAPD) merkers het twee diagnostiese nukleotiedbasis-areas vir VVG 0425 gelewer, maar pogings om NKAA-merkers uit hierdie geamplifiseerde nukleotiedbasis-areas te onwikkel was onsuksesvol. In teenstelling hiermee het ‘n inter-retrotransposon geamplifiseerde polimorfisme (IRAP) vingerafdrukmetode die ontwikkeling van ‘n multipleks IR-NKAA polimerase kettingreaksiemetode moontlik gemaak wat die VVG 0421-, VVG 0425- en ELV 4-isolate as ’n groep aangedui het. Vingerafdruktoetsing en NKAA-merkertoetsing van die 88 ongekaraktariseerde F. oxysporum isolate van Suid-Afrika (65 Focep en 23 nie-patogenies) het bevestig dat VVG 0425 die hoof VVG in Suid-Afrika is wat met volwasse bolle geassosieer word, aangesien 63 van die Focep-isolate die molekulêre eienskappe van VVG 0425 gehad het. Die derde doel van die studie was om vas te stel of saad en saailinge inokulumbronne van Focep is, en of dieselfde genotipe (VVG 0425) wat op volwasse bolle dominant is, waargeneem kon word op hierdie bronne. Focep-isolate is verkry van sewe van die 13 uiesaadlotte asook van uiesaailinge wat in al vyf uiesaailingkwekerye in die Wes-Kaap versamel is. Focep-saailinginfeksie was meer as dubbel in die 14-week groeistadium as wat dit in die 6-week stadium was. Saadinfeksies deur Focep was laag, maar die saadgedraagde aard van Focep is bevestig deur aan te toon dat ’n Focep-transformant wat met ‘n groen fluoreserende proteïen geëtiketeer is, van geïnfekteerde grond na uiesaad oorgedra kon word via die uiebolle en -saadstele. Dit is dus duidelik dat kommersiële saad en saailinge as inokulumbronne van Focep dien. Die Focep-genotipes op saad en saailinge verskil egter van dié in volwasse bolle en is nie deur VVG 0425 gedomineer nie. Verder was die meeste (≤ 60%) saad- en saailingisolate matig virulent, in teenstelling met die meestal hoogs virulente isolate uit volwasse bolle.