Plant roots are associated with a diverse microbial community of bacteria and fungi. The root microbiome communities associate with the root surface or penetrate the superficial layers of root tissues. It is of high agronomic interest to characterize root microbiomes and identify microbes with beneficial effects on plant defense against pathogens. Soybean taproot decline (TRD) is an emerging soil-borne disease caused by a fungus classified as Xylaria necrophora, challenging to control and with devastating effects on yield. Our group has initiated a study of the soybean root microbiome with three primary experiments: (I) perform a comparative study of root microbiomes and mycobiomes from healthy and TRD-affected soybean, (II) isolate, screen and (III) identify soybean root bacterial endophytes with anti-X. necrophora and anti-TRD properties. Our long-term objective is to discover and implement microbiome-based management techniques that enhance the health and yield of soybeans.

Unraveling soil microbial ecology is essential for improving sustainable agricultural productivity. Community-based studies revolutionized this field in the last decades, but much is yet to be disclosed. This thesis proposed an approach to increase the resolution of such studies by combining 16S rDNA high-throughput sequencing and population genomics, aiming to further explore the differences pointed by community analyses, as well as to overcome the limitations of using operational taxonomic units (OTUs) as ecological entities, and to introduce the evolutionary thinking in microbial ecology. Our main goal was to understand the features that make bacteria able to colonize sugarcane rhizosphere or live saprophytically in bulk soil. Rhizosphere and bulk soil are contrasting habitats for microbial life as they are highly distinct in its physical, chemical and consequently biological characteristics. Our results indicated that sugarcane shapes the rhizosphere microbiome and metabolism of D-galacturonic acid is a key function for colonizing this niche. Among the taxa prevailing in the rhizosphere, Pseudomonas genus was targeted for a more detailed study considering its known attributes for plant growth promotion. Seventy-six fluorescent Pseudomonas spp. were isolated and submitted to whole genome sequencing (WGS). A comparative genomic analysis was performed between populations from rhizosphere and bulk soil. Phylogenetic analyses classified the isolates in the P. fluorescens (57) or P. putida (19) groups. Twelve putative new species and two new proposed P. fluorescens subgroups were found in the prospected tropical soil. Comparative genomics revealed that phosphatases or xylose-utilization genes were significantly enriched in the rhizosphere and bulk soil populations of the P. fluorescens group, respectively. D-galactonate catabolism was higher in the rhizosphere population of the P. putida group based on both genotypic and phenotypic results. Growth in D-xylose was further explored using genetic modified strains and confirmed that this sugar is more used by members of the bulk soil than the rhizosphere population of the P. fluorescens group, a pattern also observed in the bulk soil microbiome. In summary, these findings constitute a step forward in understanding the ecology of rhizosphere and bulk soil bacteria, by overcoming some limitations of community-based analyses and showing genomic differences between bacterial populations of these habitats. / Desvendar a ecologia microbiana do solo é essencial para aumentar a produtividade agrícola sustentável. Estudos baseados em comunidades revolucionaram esse campo nas últimas décadas, mas ainda há muito a ser revelado. Esta tese propôs uma abordagem para aumentar a resolução desses estudos, combinando sequenciamento em larga escala de rDNA 16S e genômica populacional, com o objetivo de explorar mais a fundo as diferenças apontadas por análises de comunidades, assim como superar as limitações do uso de unidades taxonômicas operacionais (UTOs) como entidades ecológicas e introduzir o pensamento evolutivo na ecologia microbiana. Nossa principal meta foi entender as características que tornam as bactérias hábeis em colonizar a rizosfera de cana-de-açúcar ou viver no solo saprofiticamente. Rizosfera e solo são hábitats contrastantes para a vida microbiana, já que são altamente distintos em suas características físicas, químicas e, consequentemente, biológicas. Nossos resultados indicaram que a cana-de-açúcar modifica o microbioma da rizosfera e o metabolismo do ácido D-galacturônico é uma função chave para colonizar este nicho. Dentre os táxons que prevalecem na rizosfera, o gênero Pseudomonas foi escolhido para um estudo mais detalhado, considerando os seus atributos de promoção de crescimento de plantas. Setenta e seis Pseudomonas spp. fluorescentes foram isoladas e submetidas ao sequenciamento do genoma. Uma análise de genômica comparativa foi realizada entre as populações obtidas do solo e rizosfera. As análises filogenéticas classificaram os isolados nos grupos P. fluorescens (57) ou P. putida (19). Doze prováveis novas espécies e dois novos subgrupos propostos de P. fluorescens foram encontrados no solo tropical prospectado. A genômica comparativa revelou que genes de fosfatases e de uso de xilose foram significativamente enriquecidos nas populações da rizosfera e solo do grupo P. fluorescens, respectivamente. O catabolismo do ácido D-galactônico foi maior na população da rizosfera do grupo P. putida, baseado tanto em resultados genotípicos quanto fenotípicos. O crescimento em D-xilose foi mais explorado usando linhagens geneticamente modificadas e confirmou que este açúcar é mais utilizado por membros da população do solo do que da rizosfera no grupo P. fluorescens, um padrão também observado no microbioma do solo. Em resumo, essas descobertas constituem um passo adiante no entendimento da ecologia bacteriana do solo e rizosfera, por superar algumas limitações de análises de comunidades e mostrar diferenças genômicas entre populações bacterianas destes hábitats.

Unraveling soil microbial ecology is essential for improving sustainable agricultural productivity. Community-based studies revolutionized this field in the last decades, but much is yet to be disclosed. This thesis proposed an approach to increase the resolution of such studies by combining 16S rDNA high-throughput sequencing and population genomics, aiming to further explore the differences pointed by community analyses, as well as to overcome the limitations of using operational taxonomic units (OTUs) as ecological entities, and to introduce the evolutionary thinking in microbial ecology. Our main goal was to understand the features that make bacteria able to colonize sugarcane rhizosphere or live saprophytically in bulk soil. Rhizosphere and bulk soil are contrasting habitats for microbial life as they are highly distinct in its physical, chemical and consequently biological characteristics. Our results indicated that sugarcane shapes the rhizosphere microbiome and metabolism of D-galacturonic acid is a key function for colonizing this niche. Among the taxa prevailing in the rhizosphere, Pseudomonas genus was targeted for a more detailed study considering its known attributes for plant growth promotion. Seventy-six fluorescent Pseudomonas spp. were isolated and submitted to whole genome sequencing (WGS). A comparative genomic analysis was performed between populations from rhizosphere and bulk soil. Phylogenetic analyses classified the isolates in the P. fluorescens (57) or P. putida (19) groups. Twelve putative new species and two new proposed P. fluorescens subgroups were found in the prospected tropical soil. Comparative genomics revealed that phosphatases or xylose-utilization genes were significantly enriched in the rhizosphere and bulk soil populations of the P. fluorescens group, respectively. D-galactonate catabolism was higher in the rhizosphere population of the P. putida group based on both genotypic and phenotypic results. Growth in D-xylose was further explored using genetic modified strains and confirmed that this sugar is more used by members of the bulk soil than the rhizosphere population of the P. fluorescens group, a pattern also observed in the bulk soil microbiome. In summary, these findings constitute a step forward in understanding the ecology of rhizosphere and bulk soil bacteria, by overcoming some limitations of community-based analyses and showing genomic differences between bacterial populations of these habitats. / Desvendar a ecologia microbiana do solo é essencial para aumentar a produtividade agrícola sustentável. Estudos baseados em comunidades revolucionaram esse campo nas últimas décadas, mas ainda há muito a ser revelado. Esta tese propôs uma abordagem para aumentar a resolução desses estudos, combinando sequenciamento em larga escala de rDNA 16S e genômica populacional, com o objetivo de explorar mais a fundo as diferenças apontadas por análises de comunidades, assim como superar as limitações do uso de unidades taxonômicas operacionais (UTOs) como entidades ecológicas e introduzir o pensamento evolutivo na ecologia microbiana. Nossa principal meta foi entender as características que tornam as bactérias hábeis em colonizar a rizosfera de cana-de-açúcar ou viver no solo saprofiticamente. Rizosfera e solo são hábitats contrastantes para a vida microbiana, já que são altamente distintos em suas características físicas, químicas e, consequentemente, biológicas. Nossos resultados indicaram que a cana-de-açúcar modifica o microbioma da rizosfera e o metabolismo do ácido D-galacturônico é uma função chave para colonizar este nicho. Dentre os táxons que prevalecem na rizosfera, o gênero Pseudomonas foi escolhido para um estudo mais detalhado, considerando os seus atributos de promoção de crescimento de plantas. Setenta e seis Pseudomonas spp. fluorescentes foram isoladas e submetidas ao sequenciamento do genoma. Uma análise de genômica comparativa foi realizada entre as populações obtidas do solo e rizosfera. As análises filogenéticas classificaram os isolados nos grupos P. fluorescens (57) ou P. putida (19). Doze prováveis novas espécies e dois novos subgrupos propostos de P. fluorescens foram encontrados no solo tropical prospectado. A genômica comparativa revelou que genes de fosfatases e de uso de xilose foram significativamente enriquecidos nas populações da rizosfera e solo do grupo P. fluorescens, respectivamente. O catabolismo do ácido D-galactônico foi maior na população da rizosfera do grupo P. putida, baseado tanto em resultados genotípicos quanto fenotípicos. O crescimento em D-xilose foi mais explorado usando linhagens geneticamente modificadas e confirmou que este açúcar é mais utilizado por membros da população do solo do que da rizosfera no grupo P. fluorescens, um padrão também observado no microbioma do solo. Em resumo, essas descobertas constituem um passo adiante no entendimento da ecologia bacteriana do solo e rizosfera, por superar algumas limitações de análises de comunidades e mostrar diferenças genômicas entre populações bacterianas destes hábitats.

<p>  </p> <p>Plant roots form an intimate relationship with a diversity of soil microorganisms. Some soil-borne microbes cause harmful diseases on crops, but others promote plant growth and enhance host resilience against stressors. Beneficial bacteria have a high potential as a strategy for sustainable agricultural management, many of which have been recognized and commercialized for improving crop growth. Unfortunately, field inoculants of beneficial bacteria often give inconsistent results due to various environmental factors hindering their beneficial properties. Improving crop production utilizing beneficial bacteria requires two approaches: 1) breeding for crops with the enhanced association for beneficial bacteria and 2) improving formulation methods for producing more potent microbial products. To contribute to these goals, we address three critical questions utilizing the tomato root microbiome as a model system. First, we asked how beneficial root-associated bacteria could be efficiently identified. We developed a strategy to select beneficial bacteria from a novel collection of 183 bacterial endophytes isolated from roots of two field-grown tomato species. The results suggest that isolates with similar traits impact plant growth at the same levels, regardless of their taxonomic classification or host origin. Next, we asked whether host genetics contribute to the root microbiome assembly and response to beneficial microbes. An assessment of the root microbiome profile and plant binary interaction experiments suggested the role of host genetics in influencing root recruitment and response to beneficial bacteria. Subsequently, we asked whether root-associated bacteria induce physiological changes in root tissues in the host. We identified two isolates from our bacterial endophyte collection that significantly promoted the growth of tomato genotype H7996 (<em>Solanum lycopersicum</em>). Plant-binary interaction experiments suggested a significant increase of cell wall lignification in the root vasculature starting 96-hour post-inoculation with beneficial bacteria. Additional studies are needed to uncover a possible correlation between the induced vasculature lignification and the growth-promoting effects of the two isolates on H7996. Altogether, our findings highlight the multi-faceted role of root-associated bacteria in promoting plant growth and support the development of crop improvement strategies in optimizing host association with soil bacteria.</p>