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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

<u>Erwinia carotovora</u> subsp. <u>carotovora</u> (Ecc) strain EC14, a Gram-negative bacterium, causes soft rot on several crops, including potato. Maceration of potato tuber tissue is caused by secreted pectolytic enzymes. Other cell-degrading enzymes may also have roles in pathogenesis, including cellulases, phospholipases, and protease(s). The objectives of this research were to (1) characterize Ecc extracellular protease (Prt) and (2) elucidate its role in potato soft rot. A gene encoding a Prt, <u>prt</u>1, was cloned from cosmid pCA7 containing Ecc genomic DNA into plasmid pSK1. <u>Escherichia coli</u> transformed with pSK1 or pSK23, a subclone of pSK1, produced intracellularly a 38 kDa Prt with the same pI (4.8) as the secreted Ecc Prt. Prt1 activity produced by <u>E.coli</u>/pSK23 was inhibited by phenanthroline, which inhibits Zn-metalloproteases, but not by Ecc intracellular proteins. Analysis of deletion mutants indicated a 1.2 kb region necessary for Prt1 production. Sequencing of the pSK1 insert revealed a 1,041 bp open reading frame (ORF1) corresponding to the prtl region. ORF1 encodes a putative polypeptide of 347 amino acids with a total molecular mass of 38.8 kDa. The location of the prt1 promoter was determined to be 173 to 1,173 bp upstream from ORF1 by constructing transcriptional fusions to lacZ in plasmid pCD267. Primer extension revealed the start of prt1 mRNA 205 bp upstream of ORF1. The deduced amino acid sequence of the prt1 was compared to other proteases; it is similar to several bacterial Zn-metalloproteases. Prt1 production by Ecc was not observed during growth in rich broth; however, Northern analysis showed prt] mRNA accumulation in Ecc grown in planta. The role of prt1 in soft rot was determined by constructing a Prt1-deficient Ecc; prt1 insertionally inactivated by a kanamycin resistance gene was used to replace wildtype prt11 in the Ecc genome by homologous recombination. This mutant (L-957) had approximately 60 to 80% reduced Prt activity suggesting the presence of a second Prt (Prt2). Prt2 was purified from Ecc culture supernatant. This protease, also a metalloprotease, has a molecular mass of 45 kDa and pI of 4.8. Its amino terminal sequence had Significant sequence identity to metalloproteases from Erwinia chrysanthemi and Serratia marcescens, but not to Prt1. Further, unlike Prt1i, Prt2 was inhibited by Ecc intracellular proteins. The effect of proteases in potato tuber maceration was measured using L-957 and L-763, a Tn5 transposon mutant constructed previously. L-763 had no extracellular protease activity and may have been mutated in a regulatory region. Both mutants macerated significantly less tuber tissue than the wildtype Ecc. Reduced maceration of L-957 and L-763 was correlated with slower in planta growth. This suggests Prt1 production provides a nutritional advantage for Ecc growth on potato. / Ph. D.

Os polímeros sintéticos compõem cerca de 20% do lixo urbano no Brasil. Além da não biodegradabilidade, formam uma camada impermeabilizante que impede a passagem de líquidos e gases originados no apodrecimento dos detritos, retardando a estabilização da matéria orgânica. A exploração da madeira produz uma grande quantidade de rejeitos que não são inteiramente aproveitados para gerar energia ou outros produtos, e acarreta sérios problemas ambientais. A finalidade deste trabalho é a produção de compósitos biodegradáveis reunindo rejeitos de madeira e um polímero biodegradável - o poli (álcool vinílico) [PVA]. Para facilitar a degradação do PVA, este foi modificado por anidrido ftálico (AF). A modificação foi estudada através de tempo de gelatinização, calorimetria exploratória diferencial (DSC) e análise termomecânica dinãmica (DMTA). Resíduos da madeira Sugi (Criptomeria japonica) foram refinados até a obtenção de uma farinha com partículas menores que 63 &#181; m. Esta farinha foi adicionada ao meio de reação AF/PVA. As proporções de AF e PVA, bem como os parâmetros utilizados na prensagem foram determinados segundo um planejamento estatístico fatorial. Os compósitos foram moldados a quente (180 C e 50 MPa). Variando-se a proporção AF/PVA, compósitos com valores de módulo de elasticidade (MOE) de &#732;10 GPa e módulo de ruptura (MOR) de &#732;90 MPa na flexão foram obtidos. Os valores são inferiores aos apresentados pelo polímero puro, seco e sem plasticizante (acima de 152 MPa), porém superiores às placas de madeira reconstituída de MDF e OSB, disponíveis comercialmente, que apresentam valores de MOR em torno de 49 MPa. A degradação por microorganismos foi avaliada pelo ensaio de soterramento utilizando uma adaptação do método para avaliar a resistência de materiais lignocelulósicos aos fungos da podridão mole (Publicação IPT No. 1157 D5). Os fungos da podridão mole que ocorreram naturalmente nos compósitos foram isolados e identificados segundo a técnica do microcultivo. O gênero mais frequente foi o celulolítico Trichoderma spp. e o mais degradador foi o Chaetomium spp. As mudanças na estrutura e na morfologia dos compósitos foram estudadas através de espectroscopia de infravermelho (IR) e microscopia eletrônica de varredura (MEV). As perdas de massa e das propriedades mecânicas foram monitoradas em intervalos pré-estabelecidos. Compósitos com altas concentrações de AF apresentaram biodegradabilidades superiores à da madeira maciça de Pinus sp. e levemente inferiores à da madeira de Eucalyptus grandis, utilizadas como referências. A biodegradabilidade se deve à facilidade dos grupos ésteres, dos ftalatos de PVA e de celulose, de serem hidrolizados e de regenerar o ácido ftálico. Mesmo sob hidrólise enzimática, a estrutura PVA-AF-celulose, que forma ligações cruzadas entre microfibrilas de celulose, não se desintegra, conservando as propriedades mecânicas por muito tempo. Estas se mantêm superiores, ao término de 180 dias de exposição, às da madeira maciça de Sugi antes de entrar no processo de biodegradação. Uma correlação entre a estrutura, as propriedades físicas e mecânicas, e a taxa de biodegradação dos compósitos de rejeitos de madeira-PVA foi estabelecida. / Synthetic polymers constitute around 20% of urban solid waste in Brazil. Besides being non-biodegradable, they form an impermeable barrier that prevents the liberation of liquids and gas originated in the waste deterioration, retarding organic matter stabilization. The wood industry produces large amounts of waste wood which is not entirely utilized to produce energy or other products, and it brings several environmental problems. The aim of this study is the production of an environmentally friendly wood-based product composed of waste wood and poly (vinyl alcohol) [PVA]. PVA is the most widely produced water soluble and biodegradable synthetic polymer worldwide. However, PVA degradation in aqueous and soil environments has proved to be quite slow under unadapted conditions. To accelerate its degradation, the PVA has been modified by phthalic anhydride (AF). These modifications have been studied by means of gelation time, Differential Scanning Calorimetry (DSC) and (Dynamic Mechanical Thermal Analysis (DMTA). Sugi (Criptomeria japonica) flour (particles size &#8249; 63 &#181;m) has been obtained by milling waste samples. Wood flour has been added into AF/PVA reaction medium. AF and PVA ratios and pressing conditions have been set by factorial design. The final pressing temperature and pressure have been set as 180 &#176;C and 50MPa respectively. Varying AF/PVA ratios, composites presenting modulus of elasticity (MOE) values of &#732;10GPa and modulus of rupture (MOR) of &#732;90 MPa have been obtained. The MOR values are lower than that presented by the pure, dry, no plasticized PVA (higher than 152 MPa), but they are higher than commercial MDF (medium density fiberboard) and OSB (oriented strand board) of around 49 MPa. Degradation by microorganisms has been performed by soil burial test (method adapted from IPT Edition No. 1157 D5). Naturally occurring soft rot fungi have been isolated and identified according to micro cultivation techniques. Most frequent genus has been the cellulolytic Trichoderma spp. and most degrader has been Chaetomium spp. Changes in the composites microstructures and morphology throughout the biodegradation process have been studied by IR and SEM and decreasing in the mechanical properties monitored. The weight Ioss shown by composites with high AF concentration has been higher than the softwood Pinus sp. and comparable to the hardwood Eucalyptus grandis, utilized as witnesses. Even under enzymatic hydrolysis, the PVA-AF-cellulose structure has been only slightly broken, preserving considerable mechanical properties that remain superior to solid Sugi before entering any biodegradation process, even after 180 days of exposure. A correlation has been established between the structure, physical and mechanical properties and biodegradation rate of waste wood-PVA composites