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Investigação Sobre O Efeito Do Sistema De Cultivo Na Composição Da Microbiota Da Cana- De-Açúcar

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Investigação Sobre O Efeito Do Sistema De Cultivo Na Composição Da Microbiota Da Cana- De-Açúcar UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL INVESTIGAÇÃO SOBRE O EFEITO DO SISTEMA DE CULTIVO NA COMPOSIÇÃO DA MICROBIOTA DA CANA- DE-AÇÚCAR Lucas Amoroso Lopes de Carvalho Biólogo 2021 UNIVERSIDADE ESTADUAL PAULISTA - UNESP CÂMPUS DE JABOTICABAL INVESTIGAÇÃO SOBRE O EFEITO DO SISTEMA DE CULTIVO NA COMPOSIÇÃO DA MICROBIOTA DA CANA- DE-AÇÚCAR Discente: Lucas Amoroso Lopes de Carvalho Orientador: Prof. Dr. Daniel Guariz Pinheiro Dissertação apresentada à Faculdade de Ciências Agrárias e Veterinárias – UNESP, Câmpus de Jaboticabal, como parte das exigências para a obtenção do título de Mestre em Microbiologia Agropecuária 2021 DADOS CURRICULARES DO AUTOR Lucas Amoroso Lopes de Carvalho, nascido em 6 de julho de 1992, no município de Jaboticabal, São Paulo, filho de Paula Regina Amoroso Lopes de Carvalho e Gilberto Lopes de Carvalho. Graduou-se como Bacharel em Ciências Biológicas (2015-2018) pela Faculdade de Ciências Agrárias e Veterinárias (FCAV), Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP) – Câmpus de Jaboticabal, onde, sob orientação do Prof. Dr. Aureo Evangelista Santana, desenvolveu iniciação científica e trabalho de conclusão de curso (TCC), intitulado “Eritrocitograma de suínos em diferentes fases de criação no estado de São Paulo”. Em março de 2019, iniciou o curso de mestrado junto ao Programa de Pós-Graduação em Microbiologia Agropecuária, na FCAV/UNESP, sob orientação do Prof. Dr. Daniel Guariz Pinheiro, desenvolvendo o projeto intitulado “Investigação sobre o efeito do sistema de cultivo na composição da microbiota da cana-de-açúcar”, culminando no presente documento. AGRADECIMENTOS Aos meus pais, Paula e Gilberto, minha irmã Julia e minha namorada Michelle, que sempre acreditaram na minha capacidade e deram suporte para essa jornada. À Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista “Júlio de Mesquita Filho” (FCAV/Unesp) pelo suporte acadêmico e infraestrutura utilizada durante o período do mestrado. Ao professor Dr. Daniel Guariz Pinheiro, pela orientação, ensinamentos e, principalmente, pela liberdade e confiança que me foi depositada. Aos meus colegas do Laboratório de Bioinformática, Luis Guillermo, Michelli, Rafael, Maria Fernanda, André Aquino e Ana Paula, pelo convívio, momentos de descontração e auxílio nas análises. Aos docentes e funcionários do Departamento de Tecnologia, que sempre foram solícitos nos momentos em que as demandas oriundas do trabalho se fizeram necessárias. Ao Conselho do Programa de Pós-Graduação em Microbiologia Agropecuária e aos funcionários da Seção Técnica de Pós-Graduação, pela prestatividade e empenho em resolver qualquer eventual problema que tenha surgido durante os anos do mestrado. À Paulo Eduardo Garcia Junior e Olivia Garcia, proprietários das fazendas de onde os matérias vegetais utilizados foram coletados. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. Portanto, agradeço-os pelo auxílio financeiro. À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), pelo financiamento da pesquisa (no. 2017/09008-5). A qualquer outra pessoa que tenha colaborado de forma direta ou indireta para o desenvolvimento desse trabalho. Meus sinceros agradecimentos a todos! i SUMÁRIO RESUMO .............................................................................................................................. iii ABSTRACT .......................................................................................................................... iv LISTA DE ABREVIATURAS ................................................................................................. v LISTA DE TABELAS ............................................................................................................ vi LISTA DE FIGURAS ........................................................................................................... vii 1. INTRODUÇÃO .................................................................................................................. 1 2. OBJETIVOS GERAIS ........................................................................................................ 5 2.1. Objetivos específicos ................................................................................................................... 5 3. REVISÃO DE LITERATURA ............................................................................................. 6 3.1. Cana-de-açúcar: Características gerais ....................................................................................... 6 3.2. Sistema de cultivo convencional e orgânico ................................................................................ 8 3.3. Microrganismos associados ao solo e sua distribuição ............................................................. 11 3.4. Microrganismos associados às plantas ..................................................................................... 13 3.4.1. Microrganismos fitopatogênicos .............................................................................................. 14 3.4.2. Microrganismos promotores de crescimento .......................................................................... 15 3.4.3. A importância das interações microbioma-planta ................................................................... 17 3.5. Acessando as comunidades microbianas .................................................................................. 19 3.5.1. A abordagem Metataxonômica ............................................................................................... 21 3.5.2. Análises estatísticas aplicadas ao estudo do microbioma ...................................................... 22 4. MATERIAL E MÉTODOS ................................................................................................ 24 4.1. Design experimental e coleta ..................................................................................................... 24 4.2. Informações dos tratamentos ..................................................................................................... 25 4.3. Determinação dos parâmetros físico-químicos do solo ............................................................. 26 4.4. Enriquecimento das comunidades microbianas......................................................................... 27 4.5. Extração de DNA metagenômico ............................................................................................... 29 4.6. Construção das bibliotecas de Amplicons ................................................................................. 29 4.7. Sequenciamento de alto rendimento ......................................................................................... 31 4.8. Análise de dados ........................................................................................................................ 31 4.8.1. Demultiplexação ...................................................................................................................... 31 4.8.2. Controle de qualidade ............................................................................................................. 31 4.8.3. Fusão dos pares de reads (ITS) ............................................................................................. 33 4.8.4. Obtenção de Variantes de Sequencias de Amplicons (ASVs) ............................................... 34 4.8.5. Atribuição taxonômica e filtragem de ASVs ............................................................................ 35 4.9. Análise de microbiomas ............................................................................................................. 36 4.9.1. Alfa e Beta diversidade ........................................................................................................... 36 4.9.2. Microbioma essencial .............................................................................................................. 36 4.9.3. Táxons diferencialmente abundantes ..................................................................................... 37 4.9.4. Redes de correlação de coocorrência .................................................................................... 37 4.9.5. Predição funcional ................................................................................................................... 38 5. RESULTADOS ................................................................................................................ 39 5.1. Análise das propriedades edáficas ............................................................................................ 39 5.2. Sequenciamento e Processamento dos Amplicons ................................................................... 39 5.3. Medidas ecológicas e composição microbiana .......................................................................... 43 5.4. O microbioma essencial ............................................................................................................. 48 ii 5.5. Diferenças composicionais......................................................................................................... 49 5.6. Estruturação dos microbiomas ................................................................................................... 52 5.7. Diferenças das capacidades funcionais ..................................................................................... 54 6. DISCUSSÃO ..................................................................................................................
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