From a screen of 36 plant-associated strains of Burkholderia spp., we identified 24 strains that suppressed leaf and pseudobulb necrosis of orchid caused by B. gladioli. To gain insights into the mechanisms of disease suppression, we generated a draft genome sequence from one suppressive strain, TC3.4.2R3. The genome is an estimated 7.67 megabases in size, with three replicons, two chromosomes, and the plasmid pC3. Using a combination of multilocus sequence analysis and phylogenomics, we identified TC3.4.2R3 as B. seminalis, a species within the Burkholderia cepacia complex that includes opportunistic human pathogens and environmental strains. We generated and screened a library of 3,840 transposon mutants of strain TC3.4.2R3 on orchid leaves to identify genes contributing to plant disease suppression. Twelve mutants deficient in suppression of leaf necrosis were selected and the transposon insertions were mapped to eight loci. One gene is in a wcb cluster that is related to synthesis of extracellular polysaccharide, a key determinant in bacterial-host interactions in other systems, and the other seven are highly conserved among Burkholderia spp. The fundamental information developed in this study will serve as a resource for future research aiming to identify mechanisms contributing to biological control.

Título da Dissertação: Identificação de genes de Burkholderia sp. associados ao controle biológico de Pectobacterium carotovora.

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27Burkholderia species have different lifestyles establishing mutualist or pathogenic associations 28 with plants and animals. Changes in the ecological behavior of these bacteria may depend on 29 genetic variations in response to niche adaptation. Here, we studied 15 Burkholderia strains 30 isolated from different environments with respect to genetic and phenotypic traits. By 31Multilocus Sequence Analysis (MLSA) these isolates fell into 6 distinct groups. MLSA clusters 32 did not correlate with strain antibiotic sensitivity, but with the bacterial ability to produce 33 antimicrobial compounds and control orchid necrosis. Further, the B. seminalis strain 34 TC3.4.2R3, a mutualistic bacterium, was inoculated into orchid plants and the interaction with 35 the host was evaluated by analyzing the plant response and the bacterial oxidative stress 36 response in planta. TC3.4.2R3 responded to plant colonization by increasing its own growth 37 rate and by differential gene regulation upon oxidative stress caused by the plant, while 38 reducing the plant's membrane lipid peroxidation. The bacterial responses to oxidative stress 39 were recapitulated by bacterial exposure to the herbicide paraquat. We suggest that the ability 40of Burkholderia species to successfully establish in the rhizosphere correlates with genetic 41 variation, whereas traits associated with antibiotic resistance are more likely to be categorized 42 as strain specific. 43 44

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