Blackleg, caused by Leptosphaeria maculans, inflicts greatest canola yield losses when plants are infected before reaching the six-leaf growth stage. Studies were conducted to model pseudothecia maturation and ascospore dispersal to help growers make timely foliar fungicide applications. Pseudothecia maturation occurred mostly during the second half of June or in July in 2017 and 2018 in North Dakota and ascospores concentrations peaked during mid to late June in both years. A logistic regression model developed using temperature and relative humidity predicted the maturation of pseudothecia and ascospore dispersal with approximately 74% and 70% accuracy respectively. In addition, trials to evaluate the efficacy of five seed treatment fungicides were conducted under greenhouse and field conditions. All treatments reduced (P = 0.05) disease severity on seedlings in greenhouse trials, but not in field trials. Seed treatments, while a valuable tool, should not be used as the only means to manage blackleg.

Potato (Solanum tuberosum) is one of the most popular food crops grown as a substitute staple in Zimbabwe. Its production is constrained by pest and diseases which reduce the yield drastically. The blackleg / soft rot disease complex caused by Pectobacterium and Dickeya species (formerly known as Erwinia) has been identified as a potential threat to potato production. The soft rot pathogens can cause systemic and vascular infections in potatoes, which result in the development of various symptoms on the stem and tubers. The symptoms that develop on the plant are not species specific but depend on climatic conditions prevailing at the infection stage. The pathogens affect the crop at all stages of production, which include in the field, storage and in transit. The primary objectives of this study were to identify the pathogens which cause blackleg and soft rot on potatoes in Zimbabwe; document the grower’s knowledge of blackleg and soft rot diseases in Zimbabwe; evaluate cultivars grown in Zimbabwe and South Africa for tolerance to Pectobacterium and Dickeya species; determine the role of calcium in blackleg and soft rot development; and determine the genetic diversity of Pectobacterium carotovorum subsp. brasiliensis isolates from South Africa, Zimbabwe and mini-tubers imported from China. The first chapter introduces the research highlighting the importance of potatoes globally and in Zimbabwe. It discusses production constraints caused by soft rot pathogens, as well as the pathogenesis of Pectobacterium and Dickeya spp. It also summarises the research objectives and thesis outline. Chapter 2 provides an overview of published literature on morphology, pathogenicity and identification of blackleg and soft rot pathogens, epidemiology, host range, defense mechanisms in plants, host nutrition and disease management strategies. Taxonomic revisions have led to the reclassification of pectolytic Erwinia into several genera. Strains formerly described as Erwinia carotovora have been incorporated into the genus Pectobacterium, and strains classified as Erwinia chrysanthemi are now assigned to the genus Dickeya. Several species and subspecies of Pectobacterium and Dickeya have isolated from infected potatoes. In Zimbabwe only Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum have until now been identified as the causal agents of blackleg and soft rot diseases, respectively. Although other species and subspecies of Pectobacterium and Dickeya have been isolated from infected potato plants in Zimbabwe, their potential to cause diseases on potatoes should not be underestimated. In chapter 3 bacterial isolates collected from infected plants showing typical blackleg / soft rot disease symptoms were identified using biochemical and physiological methods, as well as rep-PCR, Amplified Fragment Length Polymorphism (AFLP) and single gene sequencing using two genes, viz. gyrB and recA. Amplified Fragment Length Polymorphisms (AFLPs) were used to study the genetic diversity among potato isolates from Zimbabwe, South Africa and mini-tubers imported from China. Pectobacterium and Dickeya species enter the plant through wounds and natural openings such as lenticels. When they invade the plant they colonise the vascular tissue and spaces between the thin walled parenchyma cells. They remain inside these cells as latent infections and disease symptoms develop when host resistance is impaired or when environmental conditions become conducive for disease development. Chapter 4 investigates the role of defense related enzymes, polyphenol oxidase, phenylalanine ammonia lyase, peroxidases, chlorogenic acid and total soluble phenols in host resistance to potato soft rot. The potato varieties were assayed for activity of polyphenol oxidase and phenylalanine ammonia lyase, peroxidases and concentration of chlorogenic acid and total soluble phenols in tuber tissue. The role of these four components in imparting resistance against the soft rot pathogens was also investigated. Blackleg and soft rot diseases are seed-borne and difficult to control using chemical, physical and cultural methods. Several approaches aimed at controlling blackleg and tuber rot have been studied but the degree of success has been variable. Plant nutrition is an important component of natural disease resistance. The effect of calcium soil amendments in enhancing resistance to potato soft rot pathogens was investigated in Chapter 5. A management strategy based on calcium-induced defense responses of potato to Pectobacterium and Dickeya spp. was investigated. Calcium promotes production of phenolics and strengthens the cell wall, making the plants resistant to maceration by pectolytic enzymes. In Chapter 6 a survey was conducted in nine potato growing regions of Zimbabwe using an informal structured questionnaire. The findings highlighted the distribution and impact of blackleg and soft rot diseases on the Zimbabwean potato industry. A broad spectrum of information on blackleg / soft rot disease complex was gathered and this information can help growers to make informed decisions about control strategies to apply. Each chapter in this thesis has been treated as an independent entity. Thus redundancy between chapters could not be avoided. It is my hope that the results of these studies on soft rot pathogens in Zimbabwe will contribute to a better understanding of the blackleg / soft rot disease complex that they cause. I also hope that these studies will form the basis of detailed and future investigations in epidemiology and disease control strategies. / Thesis (PhD)--University of Pretoria, 2012. / Microbiology and Plant Pathology / unrestricted

Pectobacterium carotovorum subsp. brasiliense (Pcb) is a plant pathogenic bacterium that causes blackleg and tuber soft rot disease of potato worldwide. Pectobacterium spp. are characterized by the secretion of large quantities of plant cell wall degrading enzymes. As the name indicates, Pectobacteria are pectinolytic pathogens, producing enzymes such as pectate lyase, polygalacturonase, and many others that are used to catalyse the breakdown of pectin, the main plant cell wall component. Consequently, virulence of Pectobacteria is highly reliant upon the production and secretion of macerating enzymes. Hence these bacteria are also referred to as “brute-force” pathogens. Infection and disease symptoms on plants commonly result in the development of blackleg disease, a characteristic black-like decay extending on the stems of infected potato plants. Furthermore, the infection of tubers results in the development of soft rot disease. Pcb is of particular interest in that among Pectobacterium spp. such as Pectobacterium atrosepticum (Pa), P. carotovorum subsp. carotovorum (Pcc), and P. wasabiae, Pcb strains are reported to be the most aggressive and virulent pathogens causing blackleg and soft rot disease of potato in many growing regions across the world. The fact that strains of Pcb were recently reported and isolated in South Africa has necessitated that this work be undertaken in order to characterise this newly described important pathogen of potato in regard to its phenotypic, genetic diversity, virulence and host range traits. Therefore in this work Pcb strains were subjected to multilocus phylogenetic analyses (MLSA) in order to investigate and determine whether there is any interspecies and intraspecies genetic diversity among the South African Pcb isolates. It was thus established that there is a significant genetic diversity that exists both on an interspecies and intraspecies level among Pcb isolates. As a result we sought to investigate further if the level of genetic diversity observed can be reflected in terms of the pathogen’s virulence, biochemical, phenotypic as well as host range characteristics. The results of virulence assays on potato tubers and stems indicated that Pcb strains are significantly much more virulent on potato compared to closely related Pectobacterium spp. such as Pa and Pcc. Moreover, the level of intraspecies diversity observed through phylogeny was also evident and reflected on the phenotypic, virulence and host range characteristics of the pathogen. This study also focused on investigating virulence factors employed by Pectobacterium spp. during infection. Such factors include the ability to produce and secrete of various extracellular macerating enzymes, as well as screening for the presence of virulence associated effectors and phytotoxin genes. It was of interest to observe that Pcb strains have the ability to grow and produce substrate-degrading enzymes much more rapidly compared to Pa and Pcc. This phenomenon was also observed in virulence assays where Pcb strains were noted to cause more rapid and most severe maceration symptoms on potato tubers and stems. Thus in agreement with other studies, our results suggests that Pcb is a uniquely sophisticated but diverse plant pathogen which can be considered to be one of the most aggressive causal agents of blackleg and soft rot disease of potato in South Africa. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Microbiology and Plant Pathology / Unrestricted

Chez S. tuberosum, les pathogènes bactériens Pectobacterium et Dickeya causent les maladies de la jambe noire et de la pourriture molle au champ et lors du stockage des tubercules. Outre les méthodes de prophylaxie, aucune méthode de lutte n’est efficace contre ces bactéries. La FN3PT/RD3PT mène des projets de recherche en phytopathologie et épidémiologie pour mieux comprendre les traits de vie et la physiologie de ces pathogènes, et proposer des solutions de lutte adaptées. L’objectif de ce travail était double : comparer le pouvoir pathogène de deux bactéries émergentes D. dianthicola et D. solani et étudier des stratégies de lutte biologique contre Pectobacterium et Dickeya. D’abord, deux souches isolées au champ, D. dianthicola RNS04.9 et D. solani 3337 ont été comparées au niveau de leur pouvoir pathogène sur tubercules et sur plante entière. Cette étape a nécessité la mise au point de pathosystèmes appropriés. D. dianthicola RNS04.9 apparait plus virulente que D. solani 3337 sur plante entière, alors que le contraire est observé sur tubercules. Une comparaison génomique complétée d’études fonctionnelles ont révélé l’exsitence de certains traits propres à chaque souche, notamment, le catabolisme de l’arabinose et de l’urée chez D. solani 3337 et celui du rhamnose chez D. dianthicola RNS04.9. Ensuite, un criblage d’isolats bactériens a été réalisé pour identifier des agents de lutte biologique capables d’inhiber la croissance de Dickeya et Pectobacterium. Six bactéries, des genres Pseudomonas ou Bacillus, ont été retenues. Les essais menés en serre ont montré l’efficacité d’une combinaison de trois Pseudomonas pour diminuer les symptômes de jambe noire causées par D. dianthicola et sa transmission à la descendance. La séquence du génome de ces agents de lutte biologique a été déterminée. Avec ces données, par qPCR, un maintien des agents de phytoprotection dans le sol a été observé. Enfin, un criblage de molécules chimiques a été réalisé sur la base de leur capacité anti-quorum-sensing pour réduire l’expression des facteurs de virulence chez Pectobacterium. Deux inhibiteurs du quorum-sensing ont été identifiés. En conclusion de ce travail, la possibilité de coupler différentes stratégies de lutte contre ces pathogènes pectinolytiques est discutée. / Pectobacterium and Dickeya phytopathogens are the causative agents of the blackleg and soft rot diseases on S. tuberosum, in the field or during tuber-storage. Today, no effective method permits to control these bacteria. The FN3PT / RD3PT conducts plant pathology and epidemiology researches to understand the life traits and physiology of these pathogens and propose adapted control solutions. The aim of this study was dual: to compare the virulence of two emerging pathogens D. dianthicola and D. solani, and to study biocontrol strategies directed at Pectobacterium and Dickeya. First, two strains were isolated from field samples, D. dianthicola RNS04.9 and D. solani 3337. The virulence of these strains was compared in tuber and whole plant-assays. This step required the development of appropriate pathosystems. D. dianthicola RNS04.9 appeared more virulent than D. solani 3337 on whole plant tests, while the opposite was observed on tubers tests. Genome comparisons and functional studies led to the dientifiation of some genetic traits unique to each strain such as the catabolism of arabinose and urea in D. solani 3337 and that of rhamnose in D. dianthicola RNS04.9. A screening of bacterial isolates was also performed to identify biocontrol agents capable of inhibiting the growth of Dickeya and Pectobacterium strains. Six isolates, belonging to the Pseudomonas or Bacillus genera were selected. The greenhouse trials have shown the efficacy of a combination of 3 Pseudomonas to reduce blackleg symptoms caused by D. dianthicola and its transmission to the offspring. The sequence of the genome of each biocontrol agents has been determined. With these data, the survival of biocontrol agents in the soil has been investigated by qPCR. Finally, a screening of chemical compounds was carried out on the basis of their anti-quorum sensing, i.e. their ability to quench the expression of Pectobacterium virulence factors. Two quorum-sensing inhibitors have been identified. As a conclusion to this work, opportunities to mix the various biocontrol strategies directed at pectinolytic pathogens is discussed.

Research objective: To assess species ratio of L. maculans and L. biglobosa during oilseed rape growing season and determine the response of oilseed rape plants to L. maculans and L. biglobosa infection. Experimental objectives: 1. To investigate seasonal dynamics of Leptosphaeria spp. ascospore release and investigate species composition of L. maculans and L. biglobosa ascospores in spore samples using real time PCR. 2. To assess the occurrence of L. maculans and L. biglobosa on different Brassicaceae plants (B. napus var. oleifera, B. oleracea var. capitata and B. oleracea var. italic). 3. To investigate species diversity of L. maculans and L. biglobosa in the population using molecular markers. 4. To assess dynamics of blackleg sympthoms after inoculation with L. maculans and L. biglobosa and identify differentially expressed genes in oilseed rape. / Tyrimų tikslas: nustatyti L. maculans ir L. biglobosa rūšių santykį rapsų vegetacijos metu, bei identifikuoti diferenciškai ekspresuojamus rapsų augalų genus L. maculans ir L. biglobosa infekcijos metu. Tyrimų uždaviniai: 1. Ištirti Leptosphaeria spp. aukšliasporių plitimo dėsningumus bei L. maculans ir L. biglobosa rūšių santykį grybo populiacijoje rapsų vegetacijos metu iš aukšliasporių panaudojant tikrojo laiko PGR su rūšims specifiniais pradmenimis. 2. Nustatyti L. maculans ir L. biglobosa rūšių paplitimą ant įvairių bastutinių šeimos augalų rūšių (Brassica napus var. oleifera, B. oleracea var. capitata, B. oleracea var. italica). 3. Molekulinių žymeklių pagalba įvertinti L. maculans ir L. biglobosa rūšių skirtingumą ir izoliatų giminingumą rūšies viduje. 4. Ištirti fomozės požymių dinamiką po rapsų augalų užkrėtimo L. maculans ir L. biglobosa piknosporomis (dirbtinio užkrėtimo sąlygomis šiltnamyje), bei identifikuoti diferenciškai ekspresuojamus rapsų augalų genus L. maculans ir L. biglobosa grybų piknosporomis užkrėstuose augaluose.

Descreve-se os achados epidemiológicos e clínico-patológicos das clostridioses diagnosticadas no Setor de Patologia Veterinaria da Universidade Federal do Rio Grande do Sul no período 1996-março/2010. Este estudo incluiu uma pesquisa retrospectiva nos arquivos do SPV e uma etapa prospectiva, a qual também teve o objetivo de desenvolver exames complementares específicos para cada clostridiose. As clostridioses mais prevalentes foram tétano (em equinos, bovinos, ovinos e caprinos), botulismo (em bovinos, suínos e aves) e enterotoxemia (em caprinos). Também houve casos de edema maligno em equinos, bem como de carbúnculo sintomático e hemoglobinúria em bovinos. Adicionalmente, foram coletadas amostras de soro sanguíneo de animais afetados por tétano, em diferentes estágios de evolução da doença, para subsequente inoculação em camundongos (testes de bioensaio) e comprovação da técnica no diagnóstico da enfermidade. / This study describes the epidemiological and clinicopathological findings of clostridial diseases diagnosed in the 1996-March, 2010 period in the Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (SPV_UFRGS). A retrospective survey in the files of SPV was complemented with a prospective phase, which also aimed developing complementary diagnostic tests of clostridiosis. The most prevalent clostridiosis were tetanus (in horses, cattle, sheep and goats), botulism (in cattle, pigs and birds), and enterotoxemia in goats. There also were cases of malignant edema in horses, blackleg and bacillary hemoglobinuria in cattle. In addition, blood serum samples from animals affected by tetanus on different stages of the disease evolution were applied in mice bioassay, as a complementary diagnosing test for the disease.

No description available.

Descreve-se os achados epidemiológicos e clínico-patológicos das clostridioses diagnosticadas no Setor de Patologia Veterinaria da Universidade Federal do Rio Grande do Sul no período 1996-março/2010. Este estudo incluiu uma pesquisa retrospectiva nos arquivos do SPV e uma etapa prospectiva, a qual também teve o objetivo de desenvolver exames complementares específicos para cada clostridiose. As clostridioses mais prevalentes foram tétano (em equinos, bovinos, ovinos e caprinos), botulismo (em bovinos, suínos e aves) e enterotoxemia (em caprinos). Também houve casos de edema maligno em equinos, bem como de carbúnculo sintomático e hemoglobinúria em bovinos. Adicionalmente, foram coletadas amostras de soro sanguíneo de animais afetados por tétano, em diferentes estágios de evolução da doença, para subsequente inoculação em camundongos (testes de bioensaio) e comprovação da técnica no diagnóstico da enfermidade. / This study describes the epidemiological and clinicopathological findings of clostridial diseases diagnosed in the 1996-March, 2010 period in the Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (SPV_UFRGS). A retrospective survey in the files of SPV was complemented with a prospective phase, which also aimed developing complementary diagnostic tests of clostridiosis. The most prevalent clostridiosis were tetanus (in horses, cattle, sheep and goats), botulism (in cattle, pigs and birds), and enterotoxemia in goats. There also were cases of malignant edema in horses, blackleg and bacillary hemoglobinuria in cattle. In addition, blood serum samples from animals affected by tetanus on different stages of the disease evolution were applied in mice bioassay, as a complementary diagnosing test for the disease.

Descreve-se os achados epidemiológicos e clínico-patológicos das clostridioses diagnosticadas no Setor de Patologia Veterinaria da Universidade Federal do Rio Grande do Sul no período 1996-março/2010. Este estudo incluiu uma pesquisa retrospectiva nos arquivos do SPV e uma etapa prospectiva, a qual também teve o objetivo de desenvolver exames complementares específicos para cada clostridiose. As clostridioses mais prevalentes foram tétano (em equinos, bovinos, ovinos e caprinos), botulismo (em bovinos, suínos e aves) e enterotoxemia (em caprinos). Também houve casos de edema maligno em equinos, bem como de carbúnculo sintomático e hemoglobinúria em bovinos. Adicionalmente, foram coletadas amostras de soro sanguíneo de animais afetados por tétano, em diferentes estágios de evolução da doença, para subsequente inoculação em camundongos (testes de bioensaio) e comprovação da técnica no diagnóstico da enfermidade. / This study describes the epidemiological and clinicopathological findings of clostridial diseases diagnosed in the 1996-March, 2010 period in the Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (SPV_UFRGS). A retrospective survey in the files of SPV was complemented with a prospective phase, which also aimed developing complementary diagnostic tests of clostridiosis. The most prevalent clostridiosis were tetanus (in horses, cattle, sheep and goats), botulism (in cattle, pigs and birds), and enterotoxemia in goats. There also were cases of malignant edema in horses, blackleg and bacillary hemoglobinuria in cattle. In addition, blood serum samples from animals affected by tetanus on different stages of the disease evolution were applied in mice bioassay, as a complementary diagnosing test for the disease.