Thesis (M.Sc. (Agriculture)) --University of Limpopo, 2018 / Tomato is second most cultivated crop globally and in South Africa it is planted by both commercial and smallholder farmers. However, the crop is susceptible to a number of diseases including those caused by fungal pathogens. Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici and seedling damping-off caused by Rhizoctonia solani, are known to cause serious yield loss in tomato production. Their management is mainly based on the application of synthetic fungicides and cultural practices. However, both methods have limitations which result in their inefficiency. Synthetic fungicides also have negative impact on the environment and human health. The ability of fungal pathogens to develop resistance to fungicides has also resulted in their reduced application. These challenges have led to a need to identify novel methods using plant extracts and biological control agents which can be used to manage these diseases. The objectives of this study were therefore to, firstly determine the efficacy of both plant extracts on mycelial growth of F. oxysporum f. sp. lycopersici and R. solani under laboratory conditions and secondly, to evaluate the effectiveness of both plant extracts as well as antagonistic fungi Trichoderma harzianum against Fusarium wilt and damping-off of tomato under greenhouse conditions. Food poisoning assay was used to investigate the efficacy of M. burkeana and M. oleifera extracts in vitro. Six (0, 2, 4, 6, 8, 10 g/ml) treatments were arranged in a completely randomised design and replicated four times. After 7 days of incubation at 25 °C, radial growth colony was measured. For the greenhouse xp im nt, Fusa ium wilt was t st d on cv. ‘HTX14’ as th most susc ptibl cultiva whilst seedling damping-off was t st d on cv. ‘Mon y-make ’. Aqu ous xt acts were prepared by decocting different concentrations of M. burkeana (4, 6, 8 g/ml) xiv and M. oleifera (2, 4 and 6 g/ml) in 100 ml of distilled water at 100 °C for 15 minutes then left to cool before filtering and applying as a treatment. Trichoderma harzianum as a treatment was applied 7 days after inoculating the soil-borne pathogens. In-vitro M. burkeana treatments concentrations had the highest mycelia growth suppression against both F. oxysporum f. sp. lycopersici at 10 g/ml (76 %) whilst suppression on R. solani was at 8 g/ml (71 %) relative to control. Moringa oleifera xt acts’ highest pathogen suppression for both F. oxysporum f. sp. lycopersici and R. solani were respectively 35 % and 60 % relative to control at concentration 6 g/ml. Under greenhouse conditions shoot disease severity had highest suppression at 0.6 g/ml of M. burkeana and 0.4 g/ml of M. oleifera treatment concentrations resulting to 32 and 49 % relative to control. Whereas, treatment 0.8 g/ml of M. burkeana and 0.4 g/ml of M. oleifera suppressed stem and root discoloration by 39 and 54 % respectively. Trichoderma harzianum significantly (P ≤ 0.05) reduced shoot severity and root and stem discolouration contributing the highest suppression of 49 % relative to control. In damping-off treatments, both plant extracts and T. harzianum also significantly duc d (P ≤ 0.05) pre- and post-emergence damping-off incidence with M.burkeana recording the highest suppression at 78 % followed by M. oleifera at 64 %. Trichoderma harzianum reduced incidence of damping-off by 60 % relative to untreated control on both M. burkeana and M. oleifera experiments. The results of this study showed that M. burkeana, M. oleifera extracts and T. harzianum can be highly suppressive to both tested plant diseases. However, further studies should be conducted to determine their mode of action, application method and their effect on other soil microorganisms. Keywords: Damping-off, Fusarium wilt, Plant extracts, T. harzianum, Tomato plant

Doubled haploidy (DH) is a plant breeding technique that is often utilized by plant breeders to minimize the time required to reach homozygosity in breeding lines. The first objective of this study was to compare two methods of generating DH lines in tobacco (Nicotiana tabacum L.). Inbred burley tobacco varieties TN 90LC and GR 149LC were used to produce both androgenic derived doubled haploids (ADDH) and maternally derived doubled haploids (MDDH). The relative agronomic performance of TN 90LC and GR 149 LC ADDH and MDDH lines was compared when used either as pure-line cultivars or when used for the production of the KT 204LC and TN 97LC hybrid cultivars, respectively. The ADDH method was more efficient than the MDDH method in generating large numbers of haploid plants. On average the ADDH TN 90LC population was statistically inferior to the inbred TN 90LC for several agronomic traits; this inferiority of the ADDH method was not observed in the GR 149LC populations. For both genotypes, the MDDH populations were comparable to the inbred parental genotypes. The ADDH method was inferior for TN 90LC, but several individual TN 90LC ADDH lines were equal or superior to the inbred source. The agronomic variability observed in both ADDH and MDDH lines was decreased when they were used to produce hybrid cultivars. Less variation was observed in the DH-derived hybrids KT 204LC and TN 97LC compared to the ADDH and MDDH TN90LC and GR149LC parental lines, respectively. The significant inferiority of ADDH TN 90 lines in comparison to inbred TN 90LC was not observed in the ADDH derived KT 204 population compared to KT 204LC. The second objective of this study was to compare DH Lines derived from an F1 breeding population versus DH lines derived from a segregating F2 population where plants used for DH were pre-screened for quantitatively inherited resistance to soil-borne diseases black shank (Phytophthora nicotianae) and/or Fusarium wilt (Fusarium oxysporum f. nicotianae). There was a clear difference in susceptibility to black shank between the F1 and F2 derived DH populations, both in terms of average disease incidence, and more importantly, in the percentage of individual lines displaying high disease resistance. For two different burley crosses, DH lines derived from the F1 generation were considerably more susceptible to black shank than DH lines derived from the F2 generation. No differences in the incidence of Fusarium wilt were observed between DH lines of F1 and F2 generations; this was likely due to low overall disease incidence. Although delaying the DH process in tobacco from the F1 to the F2 generation could add time to the development of homozygous breeding lines, the delay may be offset by having to screen fewer finished DH lines to identify superior lines.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-14T16:51:26Z No. of bitstreams: 1 Leonardo Tavares de Souza (1).pdf: 547012 bytes, checksum: 3ce24d8a338d375481b7eaa6393560c4 (MD5) / Made available in DSpace on 2017-03-14T16:51:26Z (GMT). No. of bitstreams: 1 Leonardo Tavares de Souza (1).pdf: 547012 bytes, checksum: 3ce24d8a338d375481b7eaa6393560c4 (MD5) Previous issue date: 2009-02-18 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici, is an important disease of tomato (Solanum lycopersicon L.) in Northeastern Brazil. Order to select genotypes with potential for use in the disease management, 60 strains were evaluated (F7 generation) from the crossing of access BHRS-2, 3 and Viradoro cultivar in relation to isolates from the physiologic races 2 and 3 of F. oxysporum f. sp. lycopersici. Seedlings 21 days old were inoculated using the method of cutting the roots and soak in the suspension of conidia of the pathogen. The evaluation was performed after 21 days, with the scale of grades ranging from 1 to 5. The genotypes were grouped into five classes of reaction. Most genotypes (73.3%) behaved as highly resistant to the race 2 isolate, while 45.0% were classified as susceptible and 28.3% as highly susceptible to the race 3 isolate. Only the L-1 strain showed high resistance reaction to both isolates. The stability of this line of resistance was evaluated on five isolates ofeach race (2 and 3). The line L-1 showed high levels of resistance to all race 2 isolates, therefore indicating high stability of resistance. However, for race 3 isolates, this strain showed three distinct classes of reaction, ranging from highly resistant to susceptible, indicating instability of resistance to this race. / A murcha-de-fusário, causada por Fusarium oxysporum f. sp. lycopersici, é uma importante doença do tomateiro (Solanum lycopersicon L.) no Nordeste brasileiro. Visando selecionar genótipos com potencial de utilização no manejo da doença, foram avaliadas 60 linhagens (geração F7) oriundas do cruzamento entre o acesso BHRS-2,3 e a cultivar Viradoro, em relação a isolados das raças fisiológicas 2 e 3 de F. oxysporum f. sp. lycopersici. Mudas com 21 dias de idade foram inoculadas pelo método do corte de raízes e imersão na suspensão de conídios do patógeno. A avaliação foi realizada após 21 dias, com o auxílio de escala de notas de 1 a 5, para agrupamento dos genótipos em cinco classes de reação. A maioria dos genótipos (73,3%) se comportou como altamente resistente ao isolado da raça 2, enquanto 45,0% foram classificados como suscetíveis e 28,3% como altamente suscetíveis ao isolado da raça 3. Somente a linhagem L-1 apresentou reação de alta resistência aos dois isolados de ambas as raças. A estabilidade da resistência dessa linhagem foi avaliada em relação a cinco isolados de cada raça (2 e 3) do patógeno. A linhagem L-1 apresentou reação de alta resistência a todos os isolados da raça 2, evidenciado estabilidade da resistência. No entanto, em relação aosisolados da raça 3, essa linhagem apresentou três classes de reação distintas, variando de altamente resistente a suscetível, indicando instabilidade da resistência à essa raça.

Fusarium oxysporum f. sp. vasinfectum (Fov) was first identified in Australia in 1993, and has since become one of the most significant threats to the country’s thriving cotton industry. The interaction between a unique Australian biotype of Fov and cotton hosts with varying susceptibilities to Fusarium wilt was studied. This research described the infection process and associated host defence mechanisms of two commercial cotton varieties after inoculation with Fov, and quantified their subsequent accumulation of antimicrobial terpenoids. / A rapid, reliable glasshouse bioassay that correlated with field resistance was developed for the study of Fusarium wilt of cotton. Detailed observations of the infection process obtained through light microscopy were used to formulate the disease cycle of Australian Fusarium wilt cotton. Using pathogen growth assays, varietal differences in root exudates and vascular tissues in the cotton hosts were documented. Root diffusate from the most susceptible cotton variety to Fusarium wilt, Siokra 1-4, contained a lipophilic compound that promoted the germination of Fov microconidia. On the other hand, a lipophilic compound present in diffusate from the least susceptible variety, Sicot 189, inhibited the growth of Fov germ tubes. / A bioassay using inoculated whole plants showed that Fov colonisation of the vascular tissues of Sicot 189 was restricted after 3 days. The basis for this inhibition was investigated further using light and transmission electron microscopy. Infection induced the reorganisation of contact cells in host vascular tissue, including an increase in cytoplasmic content and the partitioning of vacuoles, which was concurrent with the accumulation of materials in adjacent vessel lumens, via pits. Histochemical analysis indicated these globular materials secreted into the vessels were terpenoids. These structural and terpenoid responses in Siokra 1-4 and Sicot 189 were similar, however, they were more intense and rapid in the latter, less susceptible variety. The responses in Sicot 189 also corresponded to the time period that pathogen inhibition was observed. Thus, a correlation was demonstrated between the rapid and intense induction of both structural and biochemical responses with decreased susceptibility to Fusarium wilt. Detailed HPLC analysis of vascular tissues confirmed that terpenoids accumulated more rapidly and at higher concentrations in the less susceptible cotton variety. These findings provided strong evidence for the involvement of antimicrobial terpenoids in the determination of Fusarium wilt susceptibility of Australian cotton varieties. / This work represents the most complete survey to date of the interaction of Australian biotypes of Fov with cotton. These insights can contribute to future cotton breeding efforts and cultural management of Fusarium wilt in the field. Thus, each part of this study has advanced complementary facets of our understanding of Fov, and has provided a framework from which future studies on phytoalexins and other putative cotton defences can be studied.

Fusarium oxysporum f.sp. cubense Schlecht (Foc), causal agent of Fusarium wilt of banana (Panama disease), is considered to be one of the most serious threats to banana production in the world. There is no effective control measure for Fusarium wilt, except for the replacement of susceptible with resistant banana varieties. However, resistant varieties are not always acceptable to producers and local consumer markets. A greater awareness of the detrimental effect of chemicals on the environment has stimulated research on biological control of plant pathogens. The use of indigenous microorganims, such as non-pathogenic F. oxysporum and the bacterium Pseudomonas fluorescens, therefore, offers not only an environmentally safe but also an economical approach to combat Fusarium wilt of banana as part of an integrated disease management strategy. Non-pathogenic F. oxysporum and P. fluorescens isolates have previously been isolated from the root rhizosphere in disease suppressive soils. These isolates have the ability to reduce the incidence of Fusarium wilt in greenhouse pathogenicity trials. In this study we had hoped to expand on existing knowledge on the biological control of Fusarium wilt of banana with non-pathogenic endophytic F. oxysporum and P. fluorescens. Isolates that significantly suppress disease development in greenhouse trials were tested under field conditions. Physiological and histological studies were also performed to understand the modes of action of putative biological control agents. For the histological investigations, non-pathogenic F. oxysporum isolates were modified with green and red fluorescent proteins. Chapter 1 depicts a general overview of the biological control of Fusarium wilt diseases of agricultural crops. This chapter addresses the biology and pathogenesis of F. oxysporum, before strategies to control Fusarium wilt are discussed. The application of biological control organisms was analysed in terms of potentially useful organisms, where they can be isolated, and their possible modes of action. Finally, factors that influence biological control of Fusarium wilt diseases are discussed. A good source of prospective biocontrol agents is suppressive soils. In Chapter 2, non-pathogenic F. oxysporum isolates were collected from healthy banana roots in disease suppressive soil. Random Fragment Length Polymorphisms of the intergenic spacer region were then applied to group the non-pathogenic F. oxysporum isolates into genotypes, from which candidates were selected for biological control studies. The selected endophytes were then inoculated onto banana roots to determine their ability to act as biocontrol agents against Foc. The isolates that protected banana best against Fusarium wilt in the greenhouse, together with P. fluorescens WCS 417, were tested in the field to determine whether these isolates could effectively reduce disease incidence in an uncontrolled environment. The ability of non-pathogenic F. oxysporum and P. fluorescens WCS 417 to induce systemic resistance in Cavendish banana plants against Foc was investigated in Chapter 3 with the use of a split-root technique. The putative biocontrol agents were inoculated, separately and in combination, on one half of the roots in a split-root experiment, while the other half was challenged by a pathogenic isolate of Foc. Five different phenolic acids were assayed which included total soluble phenolic acids, non-conjugated (free acids) phenolic acids, ester-bound phenolic acids, glycosidebound phenolic acids and cell wall-bound phenolic acids. The knowledge gained will contribute to the understanding of how the biocontrol agents may induce defense responses in banana roots against Foc. Non-pathogenic isolates of F. oxysporum were transformed with the green fluorescent protein (GFP) and DsRed-Express genes in Chapter 4. These isolates were used to visualise their interactions with a GFP-transformed Foc isolate on the banana root in a non-destructive manner by means of confocal laser scanning microscopy (CLSM) in Chapter 5. The ability of non-pathogenic F. oxysporum and P. fluorescens WCS 417 to induce structural changes was also investigated with a split-root system using the CLSM. Antibioses as a mode of action of the two potential biocontrol agents was tested in vitro. Understanding the modes of action of non-pathogenic F. oxysporum and P. fluorescens WCS 417 are important when considering strategies for the implementation of these isolates in an integrated disease management strategy against Fusarium wilt of banana. / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

Management of Fusarium wilt of banana, one of the most important diseases of agricultural crops, is complicated and involves the consideration of factors such as the biology, epidemiology and population structure of the pathogen, and genetic resources and production practices of the crop. The development of an integrated disease management programme, therefore, is of great importance in countries where the Fusarium wilt pathogen, Fusarium oxysporum f.sp. cubense Foc, has been introduced into banaria fields, and where resistant cultivars are not acceptable to local markets. To achieve this, it is important to investigate new management strategies and to review methods that have been less successful in the past. These management practices need to be practical and affordable. Since certain cultural practices have proven to be effective, management practices that could compliment them should be considered. This thesis has attempted to investigate such practices in order to develop an integrated disease management programme for Fusarium wilt of banana. One of the most important findings of this study, was that the surface sterilant previously used to prevent the introduction of the Fusarium wilt into uninfected areas in South Africa, are not effective. The sterilants Sporekill and Prazin proved to be highly effective, and are now recommended to replace the sterilants previously used. Several fungicides reduced mycelial growth of Foc in vitro, with the OMI fungicides and Benomyl found to be the most effective. The same fungicides reduced the disease severity of Fusarium wilt in the greenhouse significantly, especially when they were applied as root dip treatments. None of the fungicides found effective against Foc have been evaluated in the field against Foc before. The next step, therefore, would be to evaluate root dip treatments combined with drench treatment in the field. Although it is expected that these fungicides might have a negative effect on the microbial populations in the soil, this has yet to be investigated. Fungicides may even weaken or stress the pathogen, making it more vulnerable for the action of an effective biocontrol agent or agents. Chemical activators are probably one of the most attractive strategies to combat Fusarium wilt of banana, since it stimulate the plants' own defence system. Banana plantlets were found to be quite sensitive to the amount and method whereby chemical activators were applied. The activator benzo-(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester induced resistance against Foc on the susceptible Williams cultivar in the greenhouse, but not in the field. In field studies, environmental conditions were much more variable than in the greenhouse, which made it difficult to evaluate the effectiveness of chemical activators. Sodium nitroprusside and a product containing the harpin protein showed promising results on the Williams and DRSI cultivars, respectively. These activators need to be considered as part of an integrated disease management programme. Since they are not directly applied to the soil, they will not have a negative effect on the microbial populations in the soil. Several Fusarium isolates had been collected from banana fields with disease suppressive soils in Kiepersol, South Africa. Most of these isolates were F. oxysporum, and with the exception of one isolate, proved to be non-pathogenic to banana plants. A PCR-based restriction fragment length polymorphism (RFLP) analysis of the intergenic spacer region of the ribosomal RNA operon grouped the non-pathogenic F. oxysporum isolates into 12 distinct genotypes. A great diversity could be seen among the non-pathogenic isolates compared to the pathogenic Foc isolates. The known-biological control agent F047 grouped with three of the South African isolates, while the one pathogenic isolate grouped with the pathogenic Foc from diseased Cavendish bananas in South Africa By using PCR-RFLPs, we were able to rapidly characterize the structure of non-pathogenic isolates of F. oxysporum in disease suppressive soils in Kiepersol. This could assist us in our search for potential biological control agents for Fusarium wilt of banana. Representative isolates from the 12 genotype groups were selected for evaluation of Fusarium wilt suppressive properties in banana. These non-pathogenic F. oxysporum isolates appeared to be good biological control candidates and was compared to known biological control agents and commercial biological control products. Fourteen of the non-pathogenic isolates, the combination of two Trichoderma strains form suppressive soils in South Africa, and two Pseudomonas fluorescens isolates were found to significantly reduce Fusarium wilt development in the greenhouse. The commercial products Patostop, B-rus and a mixture of arbuscular mycorrhizae were also found to suppress the disease severity of Foc significantly. The well-know biological control agent F047 proved to be not effective. Results concluded that two of the non-pathogenic F. oxysporum isolates and the two P. fluorescens isolates, one of which was the well-known WCS 417, were the most effective of all the agents evaluated. Since combinations of biocontrol agents may provide even more consistent and effective control than a single agent, future research will include the combination of biocontrol agents found effective in this study. It would also be of great value to determine the mode of action of these isolates, so that isolates with different modes of action could be combined to enhance the suppression effect. Biological control can be a very useful component of an integrated disease management programme, since the effective agent or agents can easily be established on tissue culture banana plantlets before they are planted in the field. AFRIKAANS : Een van seker die mees belangrikste grondgedraagte siektes in lanbou, is Fusarium verwelksiekt van piesangs. In Suid-Afrika, is die siekte verantwoordelik vir emstige verliese in die piesang produksie. Aangesien daar geen weerstandbiedende kultivars beskikbaar is wat deur die mark aanvaar word nie, is dit van kardinale belang dat 'n geintegreerde siekte beheer program vir Suid-Afrika ontwikkel word. Voordat so 'n program saamgestel kan word, is dit belangrik dat verskeie faktore aangaande die patogeen en piesang poduksiepraktyke in ag geneem moet word. Beheermaatreëls moet prakties en bekostigbaar wees, en moet die reeds bestaande praktyke kan bevoordeel. Studies wat in hierdie tesis aangebied word, oorweeg beheermaatreëls wat gekombineer kan word met die huidige praktyke, nadat vorige praktyke ook in ag geneem is. Daar word gesoek na nuwe meer doeltreffende en ekonomiese metodes om siektes te beheer. Metodes wat doeltreffend aangewend kan word om die voorkoms van die siekte te vermirider. In vitro en in vivo studies het getoon dat die DMI swamdoders en Benomil die groei van die patogeen en die ontwikkeling van Fusarium verwelksiekte die meeste onderdruk. Die beste resultate is in die glashuis gevind nadat die wortels van plante in die middels geweek is. Positiewe resultate is ook verkry met die grondtoediening van Benomil 'n week nadat plante geplant is in Foc geïnfekteerde grond. Die chemiese beheer van Fusarium verwelsiekte kan verder ondersoek word deur die effek van die grondtoedienings en wortelbehandelings in die veld te ondersoek. Daar word egter verwag dat die swamdoders moontlik 'n negatiewe uitwerking op die mikrobiese aktiwiteit in die grond kan veroorsaak. Die gebruik van effektiewe ontsmettingmiddels is uiters belangrik vir die voorkomende beheer van Fusarium verwelkdiekte op piesangs. Die ontsmettingmiddel, koper oxichloried, wat tot onlangs in Suid Afrika gebruik was, is ondoeltreffend gevind vir ontsmettingsdoeleindes. Prazin en Sporekill, twee omgewingsvriendelike middels, is baie effektief gevind en word dus aanbeveel vir die ontsetting van voertuie, skoene en veld toerusing. Chemiese plant aktiveerders stimuleer plante om hulleself te beskerm deur middel van weerstandsmeganisms. Piesang plante het sensitiwiteit getoon toonoor die konsentrasie en die toedieningsmetode van hierdie chemiese aktiveerders. In die glashuisproewe het die aktiveerder benzo-(1,2,3)thiadiazole-7-carbothioic suur S-metiel ester weerstand gestimuleer in die Williams kultivar. As gevolg van veranderende toestande in die veld was dit moeiliker om die chemiese aktiveerders se werking te evalueer. Nogtans het die middels natrium nitroprussied en 'n produk wat die protein harpin bevat die voorkoms van siekte op die Williams en DRS 1 plante verlaag. Chemiese aktiveerders behoort sterk oorweeg te word as deel van 'n geintegreerde beheer program, aangesien chemiese aktiveerders nie direk tot die grond aangewend word nie, en geen negatiewe uitwerking op die natuurlike mikrobiese populasies in die grond uitoefen nie. Verskeie Fusarium isolate is geisoleer vanuit siekte onderdukkende gronde in die Kiepersol area van Suid-Afrika. Die meeste van die isolate is geidentifiseer as F. oxsysporum. 'n PKR-gebaseerde restriksie fragment lengte polimorfisme (RFLP) ontleding van die "intergenic spacer region" van die ribosomale DNS operon het die niepatogeniese F. oxysporum isolate in 12 verskillende genotypes opgedeel. 'n Groot diversiteit was sigbaar onder die nie-patogeniese isolate in vergelyking met die patogeniese foc isolate. Die bekende beheer agent, Fo47 het gegroepeer saam met drie van die Suid Afrikaanse nie-patogene. Hierdie tegniek het ons in staat gestel om die nie-patogeniese populasie van onderdrukkende gronde in Kiepersol vinnig te karakteriseer en potentiele biologiese agente te identifiseer. Verteenwoordigende isolate van die 12 genotipiese groepe wat geidnetifiseer is, is geselekteer vir verdere evaluasie. Dit is gevind dat die isolate goeie kandidate vir moontlike bio-beheer agente maak. Die onderdrukkingsvermoe van die nie-patogene is vergelyk met die van bekende bio-beheer agente en komersiele produkte wat beskikbaar is. Veertien van die nie-patogene, die kombinasie van twee Trichoderma spp., en twee Pseudomonas fluorescens isolate het die siekte ontwikkeling van Fusarium verwelking merkwaardig onderdruk in die glashuis. Die komersiele produkte Patostop®, B-rus en die kombinasie van twee mycorrhizae isolate is ook gevind om die voorkoms van siekte te verlaag. Die wel-bekende biobeheer agent Fo47 is oneffektief gevind teen Fusarium verwelksiekte van piesangs. Resultate van die studie het bewys dat twee van die nie-patogeniese F.Oxysporum isolate en twee P. Fluorescens isolate, waarvan een die welbekende WCS 417 is, uiters effektiewe beheer agente teen Foc is. Toekomstige studies sal fokus op die kombinasie van die bio-beheer agente wat die meeste potensiaal getoon het in die studie, asook hulle meganismes van werking. Biologiese beheer is van groot waarde vir 'n geïntegreerde beheer program. Dit kan maklik met bestaande beheer maatreëls gekombineer word en potensiële biologiese beheer agente kan vooraf op weefselkultuur plante in die kwekery gevestig word. / Dissertation (MSc)--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13345号 / 論農博第2888号 / 新制||農||1079(附属図書館) / 学位論文||R2||N5252(農学部図書室) / (主査)教授 奥本 裕, 教授 土井 元章, 教授 冨永 達 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Yes / The significant role of plants can be observed through the dependency of animals and humans on them. Oxygen, materials, food and the beauty of the world are contributed by plants. Climate change, the decrease in pollinators, and plant diseases are causing a significant decline in both quality and coverage ratio of the plants and crops on a global scale. In developed countries, above 80 percent of rural production is produced by sharecropping. However, due to widespread diseases in plants, yields are reported to have declined by more than a half. These diseases are identified and diagnosed by the agricultural and forestry department. Manual inspection on a large area of fields requires a huge amount of time and effort, thereby reduces the effectiveness significantly. To counter this problem, we propose an automatic disease detection and classification method in radish fields by using a camera attached to an unmanned aerial vehicle (UAV) to capture high quality images from the fields and analyze them by extracting both color and texture features, then we used K-means clustering to filter radish regions and feeds them into a fine-tuned GoogleNet to detect Fusarium wilt of radish efficiently at early stage and allow the authorities to take timely action which ensures the food safety for current and future generations. / Supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries(IPET) through Agri-Bio Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs(MAFRA) (316033-04-2-338 SB030).

A doença da bananeira \'mal-do-Panamá\', causada pelo fungo Fusarium oxysporum f. sp. cubense (Foc) é uma das doenças mais destrutivas da bananeira e é considerada uma das seis doenças economicamente mais importante da história da humanidade. Algumas cultivares resistentes, como a \'BRS Platina\', foram lançadas pela Embrapa, porém para a sustentabilidade da resistência é necessário entender os mecanismos moleculares envolvidos na resposta de resistência e defesa. O objetivo deste estudo foi caracterizar o processo de infecção pelo Foc raça 1 em três cultivares contrastantes para a resistência e analisar o padrão transcricional no início da interação. A análise histopatológica indicou que o Foc raça 1 penetra pela raízes laterais e principal, colonizando os espaços inter e intracelular do córtex nas três cultivares. Foram visualizadas, hifas \'globosas\' na cultivar suscetível \'Maçã\' com a formação de estruturas de resistência, como clamidósporos. Na cultivar resistente \'BRS Platina\', foi observado por microscopia óptica no período inicial da interação (24 horas após inoculação) a indução de respostas de defesa da planta, como formação de zona de cicatrização, e aos 15 dias após inoculação, formação de tilose, presença de cristais de oxalato de cálcio e deposição de calose. Foi utilizada a tecnologia Illumina para sequenciamento massal de RNA e abordagens de bioinformática para identificar genes diferencialmente expressos (DE) relacionados com a resposta de defesa de bananeira em interações compatíveis e incompatíveis. O sequenciamento paired-end gerou um total de 113.632.486 fragmentos (reads) com alta qualidade. Do total de reads alinhados no genoma referência (\'DH-Pahang\'), 55.555.480 alinharam-se com genes conhecidos e anotados no genoma referência, sendo utilizados para a análise DE inoculado x não inoculado, permitindo detectar 2.307 genes para as três cultivares. Os genes anotados de cada cultivar foram comparados, sendo identificados quatro genes comuns para as três cultivares, dez compartilhados entre \'Maçã\' e \'Prata-anã\', 21 compartilhados entre \'BRS Platina\' e \'Maçã\', 114 compartilhados entre \'BRS Platina\' e \'Prata-anã\', além de 75 serem exclusivos de \'Maçã\', 599 de \'BRS Platina\' e 1484 de \'Prata-anã\'. O mecanismo de resistência/defesa ao Foc em \'BRS Platina\', ocorre em nível de percepção precoce na presença do patógeno desencadeando resposta de defesa inexistente em \'Maçã\', e com cinética distinta da cultivar com resposta intermediária (\'Prata-anã\'). Dessa forma, os resultados permitiram propor um modelo da resposta de defesa/resistência ao Foc raça 1 em bananeira, baseando-se no nível de indução de genes que codificam para proteínas de reconhecimento do patógeno (receptor like kinase), fatores de transcrição (WRKY e MYB); reforço e síntese de parede celular, degradação da parede celular do fungo (quitinase e glucanases), heat shocks, enzimas antioxidantes e na resposta visualizada pela histologia na cultivar \'BRS Platina\'. Sendo assim, este trabalho fornece novas perspectivas para estudos de análise funcional, identificação e anotação de novos genes relacionados a resposta de defesa e resistência ao Foc raça 1. / The banana Panama disease, caused by fungus Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive disease of the industry, and it is considered one of the six most economically important of all times. A few cultivars, such as \'BRS Platina\', were released, but it is still necessary to understand molecular mechanisms involved in defense response and resistance. The objective of this study was to characterize the infection process by Foc in three banana cultivars contrasting for resistance to Foc and to analyze the transcriptional profile at the beginning of interaction. In this way, Foc race 1 penetrated the main and lateral roots, colonizing inter- and intracellular spaces of the root cortex in the three cultivars. Hyphae were globose in the susceptible cultivar \'Maçã\' with the formation of resilience structure, such as chlamydospores. In the resistant cultivar \'BRS Platina\', during the initial period of interaction (24 hours after inoculation), induced of plant defense responses, such as a healing zone, tylosis formation, presence of calcium oxalate and callose deposition. The Illumina technology were applied to sequence RNA, followed by bioinformatic tools to identify genes differentially expressed (DE) related to resistance and defense response in the compatible and incompatible interactions. Pair-end sequencing generated a total of 113,632,486 reads with high quality. From the total of aligned reads to the banana reference genome (\'DH-Pahang\'), 55,555,480 aligned with gene models annotated in the reference genome. The aligned contigs were analysed for DE, comparing inoculated x non-inoculated, enabling the detection of 2307 genes for the three cultivars. Each annotated gene from each cultivar was compared: four common genes to the three cultiars; 10 genes were shared between \'Maçã\' and \'Prata-anã\'; 21 shared between \'BRS Platina\' and \'Maçã\'; 114 shared between \'BRS Platina\' and \'Prata-anã\', plus 75 exclusive to \'Maçã\'; 599 exclusive to \'BRS Platina\' and 1,484 to \'Prata-anã\'. The mechanism of resistance/defense in \'BRS Platina\', level of perception occurs early in the presence of the pathogen defense response triggering nonexistent in \'Maçã\' and with kinetics distinct cultivar with intermediate response (\'Prata-anã\'). Thus, the results have provided a model of defense response/resistance to Foc race 1 in banana, based on the level of gene induction that encode recognition proteins (Receptor-like Kinase, RLK), transcription factors (WRKY and MYB), cell wall synthesis and reinforcement, degradation of fungal cell wall (chitinases and glucanases), heat shocks , proteins;anto-oxidative enzymes and visualized by histologcal in response cultivar \'BRS Platina\'. The present work offer new perspectives to functional analyses, identification and annotation of new genes related to resistance and defense response to Foc race 1.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-17T15:25:38Z No. of bitstreams: 1 Cleia Santos Cabral.pdf: 1106804 bytes, checksum: 14447df94770ff469bfc234136893b59 (MD5) / Made available in DSpace on 2017-02-17T15:25:38Z (GMT). No. of bitstreams: 1 Cleia Santos Cabral.pdf: 1106804 bytes, checksum: 14447df94770ff469bfc234136893b59 (MD5) Previous issue date: 2012-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Fusarium wilt, caused by Fusarium oxysporum f. sp. lactucae (FOLac), is an important disease of lettuce in the world. This disease was reported in Brazil, recently. From 2008 to 2011 the laboratories of Plant Pathology of Embrapa Hortaliças (Embrapa Vegetable Crops), Sakata Seeds Sudamerica and Instituto Capixaba de Pesquisa, Assistência Técnica e Extensão Rural (INCAPER) collected some FOLac isolates in states from the Southern and the Southeastern regions of Brazil. The isolates were identified as F. oxysporum by the morphological characteristics of conidia and conidiophores. Isolates were inoculated on lettuce plants, cultivars Elisa, Vera, and Red Salad Bowl, conditions in a greenhouse. Isolates were also inoculated on plants of others Asteraceae species (Cichorium endivia, Cichorium intybus, Sonchus oleraceus, Emilia sonchifolia, Bidens pilosa e Tagetes erecta) and others botanical families as (Solanum lycopersicum, Capsicum annuum, Nicotiana tabacum, Gossypium hirsutum, Phaseolus vulgaris e Ocimum basilicum). Lettuce cultivars Elisa and Vera were suscetible to all isolates while the cultivar Red Salad Bowl was resistant. All isolates were reisolated from diseased plants fulfilling the Koch‟s postulates. Others plant species were not susceptible to any isolate, proving that isolates belong to the species F. oxysporum f. sp. lactucae. The isolates were also inoculated in a differential set of cultivars comprising: „Patriot‟ (Susceptible to all races), „Costa Rica No. 4‟ (resistant to race 1) and „Banchu Red Fire‟ (resistant to race 2). Cultivars „Patriot‟ and „Banchu Red Fire‟ were susceptible while „Costa Rica No. 4‟ was resistant, confirming that all the isolates were race 1. Molecular analysis using a primer specific to race 1 isolates was performed using F. oxysporum f. sp. lycopersici (FOL) raça 3 and a non-pathogenic isolate as negative controls. DNAs of FOLac isolates were amplified by PCR and those of the negative controls were not, confirming the specificity of the primers and the presence of only the race 1 of FOLac in Brazil. In addition, it was used the Translation elongation factor 1-α region (tef-1α) for phylogenetic analysis between FOLac isolates races 1 and 2 and isolates of F. oxysporum. Comparison of the sequences obtained with the tef-1α confirmed the polyphyletic origin of the forma specialis lactucae and also showed a greater genetic variability among Brazilian isolates of FOLac race 1compared with isolates of the same race available in GenBank. After the isolates characterization, it was made a screening of 102 accessions for resistance to the isolate Fus-173 and it was selected 47 as highly resistant. After this, the selected genotypes were evaluated for the stability of resistance in three additional assays, using different FOLac race 1 isolates. In all three assays it was used a highly susceptible cultivar (Regina) as susceptible control. In the first assay, carried out in October 2011, it were used the isolates Fus-202 and Fus-205. In the second assay, carried out in November 2011, it were used the isolates Fus-219 and Fus-222. In the third assay, carried out in December 2011, it were used the isolates (Fus-207, Fus-209 e Fus-220). Inoculation was performed on 25 days old seedlings on greenhouse conditions. Seedlings were inoculated by cutting their roots and emerging them in spore suspension of pathogen. Evaluation was carries out 30 days after inoculation, using a grade scale varying from 0 (heath plants) to 4 (dead plants). Data were transformed in Disease Index (DI) submitted to a variance analysis and the media were compared by the Tukey‟s test (5%). Thirty two accessions were identified as having broad spectrum of resistance to different pathogen isolates in the four inoculation seasons. / A murcha de fusário, causada pelo fungo Fusarium oxysporum f. sp. lactucae (FOLac) é uma das doenças mais importantes da alface. Esta doença foi relatada recentemente no Brasil. Nos anos de 2008 a 2011 foram coletados isolados de FOLac nos Laboratórios de Fitopatologia da Embrapa Hortaliças, Sakata Seed Sudamerica Ltda e do Instituto Capixaba de Pesquisa, Assistência Técnica e Extensão Rural (Incaper). Estes eram provenientes de todos os estados das Regiões Sul e Sudeste do Brasil. A identificação foi feita observando-se as características morfológicas de conídios e conidióforos. Os isolados foram inoculados em plantas das cultivares Elisa, Vera e Red Salad Bowl, em condições de casa de vegetação. Os mesmos também foram inoculados em plantas de outras espécies de Asteraceae (Cichorium endivia, Cichorium intybus, Sonchus oleraceus, Emilia sonchifolia, Bidens pilosa e Tagetes erecta) e de outras famílias (Solanum lycopersicum, Capsicum annuum, Nicotiana tabacum, Gossypium hirsutum, Phaseolus vulgaris e Ocimum basilicum). Os isolados foram identificados como Fusarium oxysporum. As cultivares Elisa e Vera foram suscetíveis a todos os isolados e a Red Salad Bowl foi resistente. Efetuou-se o re-isolamento do patógeno, completando-se assim os Postulados de Koch. Nenhuma outra espécie de planta foi suscetível ao patógeno, confirmando a identificação como F. oxysporum f. sp. lactucae. Em seguida, os isolados foram avaliados quanto a sua virulência numa série de cultivares diferenciadoras de raças: Patriot (suscetível), Costa Rica No. 4 (resistente à raça 1) e Banchu Red Fire (resistente à raça 2). As cultivares Patriot e Banchu Red Fire comportaram-se como suscetíveis a todos os isolados, enquanto a cultivar Costa Rica No. 4 comportou-se como resistente. Concluiu-se que os isolados avaliados são da raça 1 de FOLac. Adicionalmente, foi feito um teste com primers específicos para a raça 1 de FOLac, utilizando como controles um isolado de F. oxysporum f. sp. lycopersici (FOL) raça 3 e um isolado não patogênico de F. oxysporum. O fragmento de DNA foi amplificado por PCR para os isolados de FOLac e não foi amplificado para o isolado de FOL e nem para o isolado não patogênico de F. oxysporum. Este resultado confirma a especificidade desse par de primers e a presença apenas da raça 1 de FOLac no Brasil. Além disso, foi utilizada a região do fator de elongação da tradução (tef-1α) para análise filogenética entre os isolados FOLac raças 1 e 2 e isolados de F. oxysporum. Comparação das seqüências obtidas com o tef-1α confirmou a origem polifilética da forma specialis lactucae e também observou-se uma maior variabilidade genética entre os isolados brasileiros de FOLac raça 1 comparados com isolados da mesma raça disponíveis no GenbanK. Posteriormente, 102 acessos (cultivares comerciais ou linhagens) foram avaliados, visando identificar fontes de resistência a FOLac e analisar a estabilidade da resistência de acessos promissores a diferentes isolados do patógeno. Inicialmente foi feita uma seleção preliminar, utilizando um isolado do patógeno (Fus-173). Em seguida, quarenta e sete acessos altamente resistentes mais uma testemunha suscetível (Regina), identificados na seleção preliminar, foram reavaliados para estabilidade da resistência ao FOLac raça 1, utilizando os isolados (Fus-202 e Fus-205) no mês de Outubro de 2011; isolados (Fus-219 e Fus-222) no mês de Novembro de 2011 e isolados (Fus-207, Fus-209 e Fus-220) no mês de Dezembro de 2011. A inoculação foi realizada em condições de casa de vegetação, pelo método de corte das raízes e imersão na suspensão de conídios do patógeno. A avaliação foi realizada após 30 dias, com auxílio de escala de notas variando de 0 (planta sadia) a 4 (planta morta). Os dados obtidos foram transformados em índice de doença (ID) e submetidos a uma análise de variância e comparação de médias pelo teste de Tukey (5%). Foram identificados trinta e dois acessos apresentando amplo espectro de resistência aos diferentes isolados do patógeno nas quatro épocas de inoculação.