Center rot of onion, caused by Pantoea ananatis, was first described in the USA, in 1997. P. ananatis is seed-borne in onions and it was suggested that it was introduced into the USA on infected seed lots from South Africa. Center rot has not been observed in South Africa and it was essential to determine if P. ananatis is present in local onion seed. Colonies resembling those of P. ananatis were isolated from four South African seed lots on PA 20, a new semi-selective medium. Pathogenicity tests demonstrated that the South African and America strains induced the same symptoms on onion. Phenotypic and genotypic analyses identified the strains from seed as P. ananatis. In 2004/2005, an unreported disease of maize, brown stalk rot, was observed on commercial fields in South Africa. The representative strains induced disease symptoms similar to those observed in the field. The phenotypic and genotyping tests showed that the strains belonged to the genus Pantoea and separated them into two groups. The first group was identified as P. ananatis. The F-AFLP genomic fingerprints generated by the second group of strains, were distinctly different from those generated by known Pantoea species. To resolve the taxonomic position of Pantoea isolated from onion and maize, sixty-seven strains were subjected to a polyphasic study. The methods used included phenotypic characterisation, genomic fingerprinting, 16S rRNA gene sequence analysis and DNA-DNA hybridisation. The results revealed that the strains belong to three different species within the genus Pantoea: P. ananatis, P. vagens and a novel species, Pantoea allii sp. nov. / Thesis (PhD (Microbiology))--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

Next generation of sequencing (NGS) technologies have taken life science research into a new era. With the rapid advances in these technologies and the associated reduction in overall costs, the sequencing and assembly of genomes have come within reach of most laboratories. Studies related to the evolution, ecology and biology of an organism now rely heavily on genomic data and obtaining a genome sequence has become an essential resource for the rapid progress and success of these studies. Pantoea ananatis is recognised as an emerging but rather unconventional pathogen capable of infecting a wide range of different hosts. Numerous plants of agricultural and economic importance including maize, rice, onion, pineapple, melon, sudan grass and Eucalyptus trees have been affected. With the outbreak of P. ananatis in a South African Eucalyptus nursery in 1998, it was realised that very little is known about this pathogen. A better understanding of the pathogenicity, metabolism and ecology of the bacterium is required to develop strategies for the control of the disease. During this study, the genome sequence of P. ananatis strain LMG 20103 was obtained using the Roche 454 technology. To aid in the assembly of this Eucalyptus pathogen’s genome sequence, the type strain of P. ananatis LMG 2665 was also sequenced using Illinima’s Genome Analyzer (GA). A draft assembly of P. ananatis LMG 20103, consisting of 117 contigs, was generated after optimization of the Newbler assembly parameters and comparison with other genome assemblies and genomes. This study demonstrated that the assembly could be completed using both in-vitro, and in-silico approaches such as contig scaffolding, gap closure with conventional PCR reactions and sequencing, manual curation and automated genome annotation. The final complete genome consisted of a 4 386 227 bp chromosome and a 317 146 bp mega-plasmid. With the complete genome sequence available, the reconstruction of metabolic network of P. ananatis LMG 20103 was attempted using two pathways reconstruction pipelines namely, Pathway Tools and Model SEED. It was found that missing metabolic reactions and incomplete pathways in the draft metabolic networks were mainly caused by incorrect gene annotations or bioinformatic errors during the automated network reconstruction. These two pipelines differed substantially in the way network reconstruction is undertaken. Performing a comparison between the two proposed networks, annotation errors could be detected and corrected. Although some improvement could be made to the predicted network further experimental data is still required to improve the accuracy of the draft metabolic network. Despite the amount of effort and cost, it is believed that the complete genome and a draft metabolic network of P. ananatis LMG 20103 will be a valuable resource for many subsequent studies to investigate the evolution and biology of this emerging plant pathogen. This information will be essential for the development of strategies to predict and control future disease outbreaks associated with this pathogen. / Dissertation (MSc)--University of Pretoria, 2012. / Microbiology and Plant Pathology / unrestricted

Made available in DSpace on 2015-03-26T13:37:41Z (GMT). No. of bitstreams: 1 texto completo.pdf: 372194 bytes, checksum: 1ce7a898936fa3c0ddc79d75753c89e2 (MD5) Previous issue date: 2009-07-27 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The main objectives of this work were to identify the causal agent of the maize White Spot, to obtain preliminary information on the disease dispersal in the field and to characterize the reaction of maize hybrids and inbreds to the disease. For the etiological studies, isolations were performed from white spot lesions the anasarca phase, resulting in the development of bacterial colonies. Maize hybrids BRS2022, BRS1010, 1D2195, BRS1040, BRS1035, BRS1031, BRS3025, BRS1030, 2B710 e P30F35 and inbred lines L3, L228-3, 521274, 521236 e 262841-1-4-1 were evaluated under natural epidemic in a randomized block design with three replications. Cultivars were planted in single row plots, separated by two rows of the resistant hybrid BRS1010. Spreader rows were formed by planting the susceptible genotype DAS657 0,5 m apart and in front of each block. Disease severity was evaluated at a weekly internal starting 60 days after planting, through a 1 to 9 scale of disease severity where 1= no disease and 9= 100% of leaf area affected. Ratings were taken at three different locations within each plot: 1, 2, 3, 4, 5 and 6 meters inoculum source. Data were used for the calculation of the area under disease progress curve (AUDPC), disease severity at 50% of epidemic development (Y50), disease severity at the end of the epidemic, and the rate of disease progress. Inoculations on the susceptible hybrid DAS657, in the greenhouse, reproduced the typical symptoms of the disease. Re-isolations from theses lesions confirmed the presence of the same bacteria isolated from the field, which identified as Pantoea ananatis, confirming previous reports on the involvement of this bacteria in the initial lesions of this disease. No disease gradient was observed based on the disease severity observed in each point of evaluation within each plot. A better distinction between the level of resistance of maize genotypes was obtained through AUDPC and Ymáx values. Maize hybrids BRS1030, BRS1035 and BRS1010 and inbreds L3, and L228-3 were the most resistant genotypes. These inbred lines may be useful in breeding programs for resistance to maize white spot. / Este trabalho objetivou confrimar o agente causal da mancha-branca do milho, obter informações preliminares sobre a dispersão do patógeno e caracterizar a reação de genótipos de milho a doença. Para o estudo etiológico, isolados foram obtidos de lesões de manchabranca em fase de anasarca, resultando em desenvolvimento de colônias bacterianas. Híbridos de milho, BRS2022, BRS1010, 1D2195, BRS1040, BRS1035, BRS1031, BRS3025, BRS1030, 2B710 e P30F35 e as linhagens L3, L228-3, 521274, 521236 e 262841-1-4-1 foram avaliados sob epidemia natural em delineamento de blocos ao acaso e três repetições. Os cultivares foram plantados em fileiras de cinco metros, separadas por uma linha do híbrido resistente BRS1010. A cortina suscetível (fonte de inóculo) formada pelo híbrido DAS657, foi plantada na parte frontal de cada bloco, afastada 0,5 m. A severidade da doença foi avaliada em intervalos semanais a partir dos 60 dias do plantio, utilizando uma escala de 1 a 9, onde: 1= sem doença e 9= 100% de área foliar afetada. As avaliações foram realizadas em 6 pontos dentro da parcela afastados 1, 2, 3, 4, 5 e 6 metros da fonte de inóculo. Os dados de severidade foram usados para o calculo da área abaixo da curva de progresso da doença (AACPD), severidade da doença na metade da epidemia (Y50), severidade da doença no final da epidemia (Ymáx), e taxa de progresso da doença. Com inoculações em híbrido suscetível DAS657, em casa de vegetação, foi possível reproduzir os sintomas típicos da doença. Reisolamento a partir dessas lesões confirmou a presença da mesma bactéria isolada do campo, identificada como Pantoea ananatis, corroborando relatos do envolvimento desta bactéria nos sintomas iniciais da doença. Não foi observada a formação de um gradiente de dispersão baseado na severidade da doença observada em cada ponto de avaliação dentro da parcela. A melhor distinção entre os níveis de resistência de genótipos de milho foi obtida pelos valores de AACPD e Ymáx. Os híbridos de milho BRS1030, BRS1035 e BRS1010 e as linhagens L3, e L228-3 foram os genótipos mais resistentes. Essas linhagens podem ser usadas em programas de melhoramento visando resistência a mancha-branca.