Microbioma da Rizosfera e seu papel na supressão de doenças em plantas Dr. Lucas William Mendes Centro de Energia Nuclear na Agricultura CENA-USP Netherlands Institute of Ecology NIOO-KNAW
microbiome The totality of microorganisms, their genomes and interactions
rhizosphere microbiome Philippot et al., 2013 Nat Rev Microbiol
rhizosphere microbiome Plant growth Plant nutrition Plant health Tolerance to stress Disease suppression Philippot et al., 2013 Nat Rev Microbiol Mendes et al., 2014 ISME J
plant breeding and domestication Productivity (Kg/ha) Stability Adaptability Plant structure Root architecture Nutritional quality Grain size Grain color Cooking time Disease resistance
plant breeding and domestication Pérez-Jaramillo et al. 2015
plant breeding and domestication Watermelon Banana Carrot Source: Genetic Literacy Project
plant breeding and domestication Corn Eggplant Cabbage Source: Genetic Literacy Project
What about microorganisms? plant breeding
hypothesis The resistant cultivar recruits specific microbial groups in the rhizosphere that help the plant to fend off the fungal infection hypothesis
study model Fusarium oxysporum Fusarium wilt IAC Milênio Resistant IAC Imperador Moderately Resistant BRS Estilo Moderately Susceptible IAC Alvorada Susceptible Resistant Susceptible
genetic resistance Resistance Pool of genes (epistatic relationship) Vascular occlusion (gel plugs) Tylosis Deposition of additional wall layers Restrict colonization of the fungi Structural Chemical Infusion of phenol and other metabolites Fusarium penetrates intact root tissue Moves inter- intracellularly Reaches the xylem vessel Mace et al., 1981 Pereira et al., 2013
Objective Assess the rhizosphere microbiome of the common beans, in order to: (i) understand the effect of soil type and host genotype in shaping the microbiome (ii) identify potential microbial groups or traits that support the plant protection against the soil pathogen Resistant Cultivar Bulk Soil Susceptible Cultivar
Mesocosm Experiments Amazonian Dark Earth Agricultural Soil 4 common bean varieties Resistant and Susceptible to F. oxysporum Common beans varieties IAC Milênio Resistant IAC Imperador Moderately Resistant methodology IAC Estilo Moderately Susceptible IAC Alvorada Susceptible Taxonomic Profile DNA Extraction Bulk Soil Rhizosphere Soil Sampling Bulk Soil Rhizosphere Functional Profile DNA Extraction Bulk Soil Rhizosphere Soil Type Amazon Dark Earth (ADE) High Microbial Diversity Mesocosm Experiments Agricultural Soil (AGR) Low Microbial Diversity 16S rrna Illumina Miseq Metagenomics Illumina Miseq DNA extraction and sequencing Integrated Analysis Bioinformatic and statistical tools
RESULTS
Community Structure -1.0 Axis2 (11.3%) 1.0 A TAXONOMY resistant P OM Sand susceptible -1.0 Axis1 (28.9%) 1.0 Soil Type (ADE x AGR) PERMANOVA Sample Type (Bulk x Rhiz) PERMANOVA Cultivar PERMANOVA -1.0 Axis2 (3.6%) 1.0 B Rs Sc Sand ADE AGR Soil Rhizo Milenio Rhizo Imperador Rhizo Estilo Rhizo Alvorada FUNCTION -1.0 Axis1 (77.9%) 1.0 Mg Ntotal Soil Type (ADE x AGR) PERMANOVA Sample Type (Bulk x Rhiz) PERMANOVA Mendes et al., 2017 The ISME Journal
Diversity taxonomic analysis A B C D Soil
phylum abundance taxonomic analysis Armatimonadetes Chlamydiae Cyanobacteria Candidatus Saccharibacteria Candidate division WPS-1 Gemmatimonadetes Deinococcus-Thermus Bacteroidetes Parcubacteria Armatimonadetes Chlamydiae Cyanobacteria Euryarchaeota Candidatus Saccharibacteria Deinococcus-Thermus Unclassified Bacteria
Amazon Dark Earth microbiome core taxonomic analysis Agricultural Soil 2.5% cultivar specific 70% core shared with all cultivars Mendes et al., 2017 The ISME Journal
functional abundance functional analysis Motility and Chemotaxis Dormancy and Sporulation DNA Metabolism Regulation and Cell Signaling Membrane Transport Mendes et al., 2017 The ISME Journal
highlights Modulation of the microbial communities soil type > rhizosphere > cultivar Diversity rhizosphere > bulk soil Diversity and Abundance *TREND* > resistant Mendes et al., 2017 The ISME Journal
Resistant x Susceptible Resistant IAC Milenio X Susceptible IAC Alvorada IAC ALVORADA grain quality X BRSMG MAJESTOSO high yield resistance to Fusarium RESISTANT IAC MILENIO
Resistant x Susceptible Community Structure 16S rrna -0.8 Axis 2 (15.2%) 1.0 ADE Soil Rs Sc P = 0.0037-1.0 Axis 1 (71.3%) 2.5-0.8 Axis 2 (22.1%) 1.0 AGR Soil Rs Sc P = 0.0035-1.5 Axis 1 (61.9%) 1.5 Mendes et al., 2017 The ISME Journal
Family Level Resistant x Susceptible
Resistant x Susceptible functional analysis Genus Level Nitrogen, Phosphorus, Postassium, Sulfur, Iron, Carbohydrates Motility and chemotaxis, secondary metabolism Chitinase Dormancy and sporulation Phenazine Stress response Disease and defense Mendes et al., 2017 The ISME Journal
Resistant x Susceptible functional analysis A Light-harvesting complexes ABC transporters Spore DNA protection Protein secretion system, Type VIII Two related proteases (ABC transport system) Fatty Acids, Lipids, and Isoprenoids Organic hydroperoxide resistance protein Heat shock Sodium Ion-Coupled Energetics Phosphate metabolism RNA processing and modification Transposable elements Flagella protein Capsular and extracellular polysacchrides CRISPs Choline bitartrate degradation, putative Rs Sc Soil Rs Sc Soil Mendes et al., 2017 The ISME Journal
co-occurrence network analysis Resistant Cultivar IAC Milenio Positive Negative
co-occurrence network analysis Susceptible Cultivar IAC Alvorada Positive Negative
co-occurrence network analysis Bulk Soil Positive Negative
co-occurrence network analysis Resistant Susceptible Bulk Soil Number of Nodes 230 241 222 Number of edges 9728 5883 9457 Modularity 12.27 4.57 18.08 Communities 9 17 9 Diameter 6 7 6 Average degree 84.59 48.82 85.19
co-occurrence network analysis Resistant Cultivar IAC Milenio Dyadobacter Acidobacteria Gp16 Comamonadaceae Positive Negative
highlights Resistant IAC Milenio X Susceptible IAC Alvorada Higher diversity Higher abundance Distinct community structutre Bacillus and Pseudomonas Phenazine, Rhamnolipids and other traits Network: Complex, Connected andd Closed
conclusion Breeding for Fox-resistance in common bean may have coselected for other unknown plant traits that support a higher abundance of specific beneficial bacterial families in the rhizosphere with functional traits that reinforce the first line of defense. Rhizosphere microbiome is the first line of defense against soilborne pathogen, by means of specific microbial groups, functional traits, diversity, abundance and network structure. Mendes et al., 2017 The ISME Journal
The Future Identifify key-groups Understand the role and interactions [Plant Genotyping] link plant genome to microbial recruitment [Molecular Breeding] selection for beneficial microbial groups
backrootsproject.wix.com/backroots the project
Acknowledgements Mendes et al. 2017. Influence of resistance breeding in common bean on rhizosphere microbiome composition and function. The ISME Journal Mendes et al. Breeding for resistance to soil-borne pathogen impacts rhizosphere microbiome functions in common bean. New Phytologist - Under review Thanks to Tsai Siu Mui CENA Mattias de Hollander - NIOO Jos M Raaijmakers NIOO Rodrigo Mendes EMBRAPA Special Thanks to Sérgio AM Carbonell Alisson F Chiorato backrootsproject.wix.com/backroots lucaswmendes@gmail.com