Atendendo solicitação do(a) autor(a), o texto completo desta tese/dissertação será disponibilizado somente a partir de 28/12/2023 At the author's request, the full text of this thesis/dissertation will not be available online until Dec. 28, 2023 JOÃO WILLIAM BOSSOLANI LIME AND PHOSPHOGYPSUM IN LONG-TERM NO-TILL: SOIL QUALITY IMPROVING CROP PHYSIOLOGY AND 15N-FERTILIZER RECOVERY IN THE SOIL-PLANT SYSTEM Botucatu 2022 JOÃO WILLIAM BOSSOLANI LIME AND PHOSPHOGYPSUM IN LONG-TERM NO-TILL: SOIL QUALITY IMPROVING CROP PHYSIOLOGY AND 15N-FERTILIZER RECOVERY IN THE SOIL-PLANT SYSTEM Thesis presented to São Paulo State University, College of Agricultural Sciences, to obtain Doctor of Philosophy degree in Agronomy (Crop Science). Adivisor: Prof. Dr. Carlos Alexandre Costa Crusciol Co-advisor: Dra. Nídia Raquel Costa Botucatu 2022 Dedico esta tese de doutorado aos meus pais João Nelson e Benedita, aos meus irmãos Bruno (in memoriam) e Ellen, e ao meu avô Nelson (in memoriam), por teram me demonstrado a verdadeira definição de amor, honestidade, dignidade, sacrifício e dedicação, que ajudaram a me tornar a pessoa que sou hoje. Que cada uma das minhas conquistas seja a realização de seus próprios sonhos. AGRADECIMENTOS Ao nosso bom Deus por sempre mostrar o caminho e a verdade, e por nos conceder o privilégio da vida (mesmo que as vezes efêmera). A minha família: Benedita José Oliveira Bossolani (mãe), João Nelson Bossolani (pai), Bruno Henrique Bossolani (In memoriam; irmão), Ellen Rita Bossolani (irmã), Nelson Bossolani (In memoriam; avô), por sempre me incentivarem a seguir adiante, rumo aos meus sonhos, e por me confortarem e serem meu alicerce nessa vida. Meu muito obrigado do fundo do meu coração. É tudo por vocês. À Mariley Fonseca, pelo amor, paciência, incentivo, companheirismo e compreensão, tornando possível a consolidação deste trabalho e da manutenção da minha sanidade mental. Agradeço à Deus por tê-la em minha vida. Agradeço imensamente ao meu orientador e amigo, Dr. Carlos Alexandre Costa Crusciol. É um exemplo ímpar de ser humano e professor. Cada passo dado durante o doutorado foi guiado pelos seus exemplos e ensinamentos. Obrigado pela confiança, incentivo e paciência durante todo este período de orientação. Manifesto aqui minha eterna gratidão pela grande contribuição em minha vida profissional e pessoal nestes anos de convivência; foi uma grande oportunidade e um privilégio para mim. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001, no período entre 01/03/2018 à 31/07/2018. Um agradecimento especial à Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) pela concessão da bolsa de estudos (de 01/09/2018 à 30/06/2022) e por todo apoio financeiro (processos: 2018/11063-7 e 2019/12764-1). Obrigado pelo incentivo a ciência. Agradeço imensamente à Universidade Estadual Paulista, Faculdade de Ciências Agronômicas, em especial aos professores e funcionários do Departamento de Produção Vegetal pela dedicação e atenção com os alunos. São exemplos de pessoas e profissionais. Gostaria de agradecer ao Instituto de Ecologia da Holanda (NIOO-KNAW), especialmente ao Departamento de Ecologia Microbiana e à Dra. Eiko E. Kuramae. Obrigado pela oportunidade ter sido seu aprendiz, por lapidar meus conhecimentos, e por me tornar um cientista melhor. Ainda, estendo meus agradecimentos ao meu amigo Marcio Leite; obrigado por todas dicas e ensinamentos, pela paciência, e pela importante amizade construída durante o intercâmbio. Um agradecimento especial ao meu amigo Luiz G. Moretti, por ter sido meu guia desde 2011. Não poderia ter encontrado pessoa melhor para dividir aprendizados, conquistas, dias bons e ruins e, claro, as “buchas” do dia-a-dia. Obrigado do fundo do meu coração. Partindo da premissa de que não chegamos a lugar algum sozinhos, gostaria de agradecer à todos meus amigos e colegas de trabalho da UNESP/FCA: José R. Portugal, Vitor A. Rodrigues, Tatiani M. Galeriani, Sirlene L. Oliveira, Gabriel O. Neves, Andressa S. Dalla Côrt, Letusa Momesso, Israel A. Filho, Gabriela F. Siqueira, Ariani Garcia, Rafael Vilela, Leila Bernart, Nídia R. Costa, Murilo de Campos, Rafael Neres, Fernanda M. Souza, Lucas M. Jacomassi, Marcela P. Oliveira, Josiane A. V. Oliveira, Letícia P. Mendonça, Isabô M. Pascoaloto, entre outros que participaram desta jornada, pessoas que certamente levarei em minhas lembranças. Peço a Deus que os abençoe em suas respectivas caminhadas por este mundo. Obrigado por toda a ajuda na condução deste trabalho e por terem tornado meus dias melhores. Aos integrantes da equipe de estagiários, pela demonstração da importância do trabalho em grupo, evidenciando que a união entre pessoas determinadas pode gerar grandes obras, além de comunhão e amizade. À banca avaliadora pela disposição e tempo dedicado a esse trabalho. Aos meus amigos de morada, Thiago B. Batista e Gustavo R. F. Oliveira. Obrigado pelos bons momentos de risada, sempre regados de aprendizado. Vocês são exemplos de como cientistas devem ter amor e zelo por aquilo que fazem. Aos meus amigos de longa data, que apesar da distância, sempre mantiveram-se presentes: Alexandre Pedrinho, Luis F. Merlotti e Flávia C. Meirelles. Meu muito obrigado por tudo. “Aqueles que se sentem satisfeitos sentam-se e nada fazem. Os insatisfeitos são os únicos benfeitores do mundo” Walter S. Landor ABSTRACT The present study is part of a long-term experiment, started in 2002, at Fazenda Experimental Lageado, belonging to the College of Agricultural Sciences (UNESP/FCA), in Botucatu (SP). Continuing the experiment, which is likely to be the only one in the State of São Paulo with such a duration (19 years), the present Thesis aimed to understand the effect of long-term surface application of lime and phosphogypsum on the soil chemical and biological attributes, root growth, plant nutrition, carbon e antioxidant metabolism, 15N-fertilizer recovery in the soil-plant system, and yield of maize intercropped with ruzigrass and soybean in succession. The field part of this study has been carried out since October 2016, when lime and/or phosphogypsum were last applied. Therefore, here we tested the following treatments: i) control (no soil amendments applied), ii) phosphogypsum alone, iii) lime alone and, iv) lime and phosphogypsum combination. Soybean sowing occurred between November and December of each year, whereas maize intercropped with ruzigrass was sown in March of each year. Our results suggested that the combination of lime and phosphogypsum proved to be effective in improving soil fertility up to 1 m depth, reflecting ideal conditions for root growth of soybean and maize intercropped with ruzigrass. As a result of these changes, crop nutrition was also improved, ensuring high photosynthetic metabolism even when periods of water scarcity occurred. In the combined application of lime and phosphogypsum, antioxidant metabolism based on free radical-consuming enzymes also increased, reducing lipid peroxidation of leaf cells. This cascading effect was reflected in the increase in grain yield of both crops. Deepening the understanding of tropical agricultural systems altered by the application of lime and phosphogypsum, our microbiological studies revealed that the joint application of lime and phosphogypsum altered the soil chemical properties in a no-till intercropped system, increasing the relative abundance of total prokaryotes (archaea and bacteria), modulating genes related to the nitrogen cycle. Finally, our results also revealed that the combined application of these inputs increases the recovery of 15N fertilizer [(15NH4)2SO4] by maize and ruzigrass in intercropping, and soybean cultivated in crop rotation, which reduced N losses to the environment. Our study highlighted the importance of using soil amendments in tropical agricultural systems, mainly in exploring the synergistic effect of lime with phosphogypsum, aiming to improve the soil chemical and microbiological quality, the nitrogen fertilizer recovery, the photosynthetic and antioxidant metabolism of plants and, consequently, the grain yield of the crops. Keywords: soil acidity; root deepening; soil microbiology; nitrogen cycle; (15NH4)2SO4. RESUMO O presente estudo faz parte de um experimento de longa duração, instalado no ano de 2002, na Fazenda Experimental Lageado, pertencente à Faculdade de Ciências Agronômicas da UNESP, em Botucatu (SP). Dando continuidade ao experimento, que provavelmente deve ser o único do Estado de São Paulo com tamanha duração (19 anos), a presente tese objetivou entender o efeito da aplicação superficial de calcário e gesso à longo prazo sobre os atributos químicos e biológicos do solo, crescimento radicular, nutrição das plantas, metabolismos do carbono e antioxidante, recuperaçao do 15N-fertilizante no sistema solo-planta, e produtividade de milho consorciado com Urochloa ruziziensis e a soja em sucessão. A parte de campo deste estudo vem sendo conduzida desde outubro de 2016, momento em que foi aplicada pela última vez o calcário e/ou gesso. Portanto, aqui testamos os seguintes tratamentos: i) controle (sem aplicação de corretivos do solo), ii) gesso, iii) calcário e, iv) combinação de calcário e gesso. As semeaduras da soja ocorreram entre novembro e dezembro de cada ano, enquanto que o milho consorciado com Urochloa ruziziensis foi semeado no mês de março de cada ano. Nossos resultados sugeriram que a combinação de calcário e gesso foi eficaz em melhorar a fertilidade do solo até 1 m de profundidade, refletindo condições ideais para o crescimento radicular de soja e milho consorciados com U. ruziziensis. Como resultado dessas mudanças, a nutrição das culturas também foi melhorada, garantindo alto metabolismo fotossintético mesmo quando ocorreram períodos de escassez hídrica. Na aplicação combinada de calcário e gesso, o metabolismo antioxidante baseado em enzimas consumidoras de radicais livres também aumentou, reduzindo a peroxidação lipídica das células foliares. Esse efeito cascata refletiu-se no aumento da produtividade de grãos de ambas as culturas. Aprofundando a compreensão dos sistemas agrícolas tropicais alterados pela aplicação de calcário e gesso, nossos estudos microbiológicos revelaram que a aplicação conjunta destes insumos alterou as propriedades químicas do solo em um sistema de plantio direto consorciado, aumentando a abundância relativa de procariontes totais (archaea e bactérias), modulando genes relacionados ao ciclo do nitrogênio. Por fim, nossos resultados também revelaram que a aplicação combinada desses insumos aumenta a recuperação do 15N fertilizante [(15NH4)2SO4] pelo milho e U. ruziziensis cultivados em consórcio, e da soja cultivada sucessão, o que reduziu fortemente as perdas de N para o meio ambiente. Nosso estudo destacou a importância do uso de corretivos do solo em sistemas agrícolas tropicais, principalmente na exploração do efeito sinérgico do calcário com o gesso, visando melhorar a qualidade química e microbiológica do solo, a recuperação de fertilizantes nitrogenados, o metabolismo fotossintético e antioxidante das plantas e, consequentemente, a produtividade de grãos das lavouras. Palavras-chave: acidez do solo; aprofundamento radicular; microbiologia do solo; ciclo do nitrogênio; (15NH4)2SO4. LISTA DE ILUSTRAÇÕES Chapter 1 – Improving soil fertility with lime and phosphogypsum enhances soybean yield and physiological characteristics Figure 1 – (A) Aerial view of the study area, which is part of a long-term experiment started in 2002. (B) Schematic representation of the cropping system, including the chronological sequences of the soil amendment applications and crops grown during the agricultural year. Red arrows indicate when soil amendment applications occurred ........................ 38 Figure 2 – Climatological water balance at Botucatu-SP, Brazil, from (A) 2002 to 2017 and during the soybean crop cycles in (B) 2017/18 and (C) 2018/19. ETc, crop evapotranspiration; ETr, real evapotranspiration............................................................................. 42 Figure 3 – Changes in soil chemical properties [(A) soil pH, (B) Ca2+, (C) Mg2+, (D) base saturation (BS), (E) Al3+ and (F) SO4 2—S] and soybean root growth [(G) root dry matter and (H) root dry matter distribution] in the different treatments [control, phosphogypsum (PG), lime (L), and lime + phosphogypsum (LPG)]. * and ** indicate statistical significance at p ≤ 0.05 and p ≤ 0.01, respectively, according to the LSD (least significant difference) test. Soil chemical properties and root growth were compared between treatments for each soil depth ............................ 52 Figure 4 – Effects of the different treatments [control, phosphogypsum (PG), lime (L), and lime + phosphogypsum (LPG)] on (A) net photosynthetic rate (A), (B) stomatal conductance (gs), (C) transpiration rate (E), (D) internal CO2 concentration (ic), (E) water use efficiency (WUE), (F) sucrose concentration, (G) Rubisco activity, and (H) Susy activity in soybean leaves. Different lowercase letters indicate significant differences between treatments, and different uppercase letters indicate significant differences between growing seasons by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) ............................................................................................... 55 Figure 5 – Effects of the different treatments [control, phosphogypsum (PG), lime (L), and lime + phosphogypsum (LPG)] on (A) hydrogen peroxide (H2O2), (B) malondialdehyde (MDA), (C) superoxide dismutase (SOD), (D) catalase (CAT), (E) ascorbate peroxidase (APX), and (F) glutathione reductase (GR)] activity in soybean leaves. Different lowercase letters indicate significant differences between treatments, and different uppercase letters indicate significant differences between growing seasons by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4)..................................................... 57 Figure 6 – Effects of the different treatments [control, phosphogypsum (PG), lime (L), and lime + phosphogypsum (LPG)] on (A) shoot dry matter, (B) grain yield and (C) crude protein in grains of soybean. Different lowercase letters indicate significant differences between treatments, and different uppercase letters indicate significant differences between growing seasons by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) .................................................... 58 Figure 7 – (A) Redundancy analysis (RDA) of the correlations of enzymes related to carbon and antioxidant metabolism with soil chemical properties. The arrows indicate correlations between factors. The canonical axes are labeled with the percentage of total variance explained (%). The significance of correlations was evaluated by a Monte Carlo permutation test, and the significant soil properties are indicated by red color (p ≤ 0.05). The colored dashed lines indicate significant clusters by permutation analysis (PERMANOVA, p ≤ 0.05). (B) Heatmap of correlations (Pearson) of soybean physiological, biochemical and agronomic parameters with soil chemical properties. Only significant correlations at p ≤ 0.05 are shown. Soil organic matter (SOM), base saturation (BS), root dry matter (RDM), chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (Total chl), carotenoids (Carot), net photosynthetic rate (A), stomatal conductance (gs), internal CO2 concentration (ic), water use efficiency (WUE), hydrogen peroxide (H2O2), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), shoot dry matter (SDM) and grain yield (GY) .......................... 60 Supplementary material Figure S1 – Effects of the different treatments [control, phosphogypsum (PG), lime (L), and lime + phosphogypsum (LPG)] on (A) Chlorophyll a, (B) chlorophyll b, (C) total chlorophyll, and (D) carotenoids concentrations in soybean leaves. Different lowercase letters indicate significant differences between treatments, and different uppercase letters indicate significant differences between growing seasons by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) .............................................................................................. 80 Chapter 2 – Long-term lime and phosphogypsum amended-soils alleviates the field drought effects on carbon and antioxidative metabolism of maize by improving soil fertility and root growth Figure 1 – Climatological water balance at Botucatu-SP, Brazil, during the maize crop cycle. ETc, crop evapotranspiration; ETr, real evapotranspiration. The arrows indicate the managing and sampling time ...................... 86 Figure 2 – Changes in soil pH (A), exchangeable calcium (Ca2+) (B) and magnesium (Mg2+) (C), base saturation (BS) (D), exchangeable aluminum (Al3+) (E) and sulfate (SO4 2--S) (F) in the soil profile as affected by surface-applied lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) treatments. Different lower-case letters for each soil depth indicate significant differences between treatments by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) ..................................................................................... 94 Figure 3 – Changes in soil organic matter (SOC), phosphorus (P), iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) at 0.0–0.2 m depth as affected by surface-applied lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) treatments. Different lower-case letters for each soil depth indicate significant differences between treatments by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) ..................................................................................... 95 Figure 4 – Root dry matter (A) and root dry matter distribution (B) in the soil profile as affected by surface-applied lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) treatments. Different lower-case letters for each soil depth indicate significant differences between treatments for each growing season by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) ............................................... 96 Figure 5 – Chlorophyll a (A), chlorophyll b (B), total chlorophyll (C), and carotenoids (D) contents in maize leaves as affected by surface-applied lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) treatments. Different lower-case letters indicate significant differences between treatments for each growing season by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) ........ 98 Figure 6 – Net photosynthesis rate–A (A), stomatal conductance–gs (B), internal CO2 concentration–ic (C), and water use efficiency–WUE (D) in maize leaves as affected by surface-applied lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) treatments. Different lower-case letters indicate significant differences between treatments for each growing season by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4)..................................................... 99 Figure 7 – Rubisco activity (A), sucrose concentration (B), and Susy activity (C) in maize leaves as affected by surface-applied lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) treatments. Different lower- case letters indicate significant differences between treatments for each growing season by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4)………………………………………100 Figure 8 – Oxidative stress [hydrogen peroxide (H2O2) (A) and malondialdehyde (MDA) (B) concentrations)] and antioxidant enzyme activities [superoxide dismutase–SOD (C), catalase–CAT (D), ascorbate peroxidase–APX (E), and glutathione reductase–GR (F)] in maize leaves as affected by surface-applied lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) treatments. Different lower-case letters indicate significant differences between treatments for each growing season by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) .................................................. 101 Figure 9 – Shoot dry matter (A) and grain yield (B) of maize as affected by surface- applied lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) treatments. Different lower-case letters indicate significant differences between treatments for each growing season by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4). Effect of soil amendments on maize development (2nd growing season = 2018) at 50 days after sowing and ear development at harvest (C) .................................................................................................. 103 Figure 10 – Redundancy analysis triplot (RDA) showing the relationship between the soil fertility × crop nutrition (A), soil fertility × crop physiology (B), and crop nutrition × crop physiology (C). The canonical axes are labeled with percentage of total variance explained (%). The arrows indicate correlations between factors. The significance of these correlations was evaluated by a Monte Carlo permutation test with 999 permutations and the significant soil properties are indicated by red color (p ≤ 0.05). The color dashed lines indicate significant clusters by permutation analysis (PERMANOVA, p ≤ 0.05). Heatmap showing the correlation coefficients (Pearson) among the soil fertility, root growth, crop nutrition, crop physiology, and agronomic parameters of maize plants (D). Only significant correlations at p ≤ 0.05 are shown. Soil organic matter (SOC), base saturation (BS), sulfate (SO4 2--S), root dry matter (RDM), chlorophyll a (Chl a), Chlorophyll b (Chl b), total chlorophyll (T. Chl), carotenoids (Carot), Net photosynthesis rate (A), stomatal conductance (gs), internal CO2 concentration (ic), water use efficiency (WUE), hydrogen peroxide (H2O2), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), shoot dry matter (SDM) and grain yield (GY) ............................................................................................... 105 Supplementary material Figure S1 – Leaf transpiration rate (E) in maize leaves as affected by surface- applied lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) treatments. Different lower-case letters indicate significant differences between treatments for each growing season by Student's t-test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) ............................................................................................. 125 Chapter 3 – Modulation of the soil microbiome by long-term ca-based soil amendments boosts soil organic carbon and physicochemical quality in a tropical no-till crop rotation system Figure 1 – Schematic graph representing the field experimental design, crop system, soil amendment application and sampling .......................... 131 Figure 2 – Soil organic carbon concentration from physic fractions (A) particulate organic carbon (POC), (B) mineral-associated organic carbon (MOC), (C) total organic carbon (TOC), and from chemical fractions (D) humic acid (C-HA), (E) fulvic acids (C-FA), (F) humin (C-HU), C- HA/TOC ratio (G), C-FA/TOC ratio (H), and C-HU/TOC ratio (I) of the long-term soil with amendments (lime; PG, phosphogypsum; LPG, lime + phosphogypsum) applied in soil surface....................................... 139 Figure 3 – Sensitivity analysis (A), and PCA of regression coefficients (B) of bacterial and fungal amplicon sequence variant (ASVs), SOM physical (SOM-PF) and chemical (SOM-CF) fractions, soil fertility, and soil physics to the environment, according to the soil amendments (B). Control (C), phosphogypsum (PG), lime (L), lime + phosphogypsum (LPG) ................................................................ 141 Figure 4 – Regression coefficients of only the significant responses of soil factors (physical and chemical fractions, soil fertility, and soil physics), bacterial and fungal communities according to the soil amendments. Coefficients are significant when their 95% confidence interval do not overlap with 0. Unclassified (un). ........................................................................ 142 Figure 5 – Similarity dendogram between regression coefficients from soil factors, bacterial and fungal communities to the soil amendments. Colors from circular plots represents the variable groups. Particulate organic carbon (POC), mineral- associated organic carbon (MOC), total organic carbon (TOC), humic acid (HA), fulvic acid (FA), humin (HU), mean weight diameter (MWD), soil bulk density (SBD), microporosity (mP), and macroporosity (MP), unclassified (un) ...................................... 145 Figure 6 – Relationship between the regression coefficient and the abundance (CLR transformed data) of top 30 most negative and most negative shifts from bacterial and fungal taxa in relation to the control treatment. Unclassified (un) ............................................................................. 147 Supplementary material and methods Figure S1 – Aboveground dry matter production of maize (A) and ruzigrass (B) cultivated in a long-term soil with amendments (lime; PG, phosphogypsum; LPG, lime + phosphogypsum) applied in soil surface ............................................................................................ 169 Chapter 4 – Long-term lime and gypsum amendment increase nitrogen fixation and decrease nitrification and denitrification gene abundances in the rhizosphere and soil in a tropical no-till intercropping system Figure 1 – Schematic graphic representing the timeline of the field experiment and the respective treatments and sample collections ........................... 181 Figure 2 – Archaeal and bacterial 16S rRNA copy numbers in the rhizospheres of maize (A, D) and ruzigrass (B, E) and in bulk soil (C, F) under different treatments (control, gypsum, lime and lime + gypsum). Different lowercase letters indicate significant differences between treatments by LSD test at p ≤ 0.05. Error bars express the standard error of the mean (n = 12) ........................................................................................... 187 Figure 3 – Ratio of the copy number of N cycle functional genes to the copy number of archaeal or bacterial 16S rRNA in the rhizospheres of maize (A, D, G, J, M) and ruzigrass (B, E, H, K, N) and in bulk soil (C, F, I, L and O) under different treatments (control, gypsum, lime and lime + gypsum). Different lowercase letters indicate significant differences between treatments by LSD test at p ≤ 0.05. Error bars express the standard error of the mean (n = 12) ................................................ 189 Figure 4 – Redundancy analysis (RDA) of the relative abundance of N cycle functional genes and soil chemical properties (A-C). The arrows indicate correlations between factors. The significance of these correlations was evaluated by a Monte Carlo permutation test and is indicated by red color (p ≤ 0.05). The dashed lines indicate significant clusters by permutation analysis (PERMANOVA, p ≤ 0.05). Heatmap of the correlation coefficients (Spearman) among the relative abundance of N cycle functional genes and soil chemical properties (D). Only significant correlations at p ≤ 0.05 are shown .................................................. 191 Figure 5 – Maize and ruzigrass leaf nitrogen content (A and B) and maize grain yield (C) under different treatments (control, gypsum, lime and lime + gypsum). Different lowercase letters indicate significant differences between treatments by LSD test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) .................................................. 193 Supplementary material Figure S1 – Rainfall and maximum and minimum air temperatures during the experimental period ........................................................................ 209 Figure S2 – Copy numbers of nifH, amoA of archaea, amoA of bacteria, nirK and nosZ in rhizospheres of maize (A, D, G, J and M), ruzigrass (B, E, H, K, N) and in bulk soil (C, F, I, L and O) under different treatments (control, gypsum, lime and lime + gypsum). Different lower-case letters indicate significant differences between treatments by LSD test at p ≤ 0.05. Error bars express the standard error of the mean (n = 12) ..................... 210 Figure S3 – Heatmap of the correlation coefficients (Spearman) between the phylogenetic marker genes (16S rRNA of bacteria and archaea) and total abundance of N-cycle functional genes. Only significant correlations at p ≤ 0.05 are shown .................................................. 211 Chapter 5 – Co–application of lime and phosphogypsum increases 15n recovery and reduces its losses by modulating soil fertility, crop growth and n cycle genes Figure 1 – Weather conditions (A), main activities during experimental period (A and B), and schematic representation of the plot, 15N–labeled fertilizer microplot, in addition to the 15N–fertilizer distribution (~3 cm from the maize row), sampling area, and sowing of soybean on the residues of maize + ruzigrass (C) ...................................................................... 216 Figure 2 – Aboveground (stover + grain) dry matter yield in the first (maize and ruzigrass) (A), and second (B; soybean) growing seasons in response to soil amendments [control, phosphogypsum (PG), lime (L), and lime + phosphogypsum (LPG)]. Different lowercase or capital letters indicate significant differences between treatments by Fisher's protected LSD test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) ............................................................................................. 225 Figure 3 – 15N recovery in each compartment (plant, soil or unrecovered) from the first (A), and second (C) growing seasons; 15N–fertilizer remaining after the first growing season (B), total 15N unrecovered (first + second growing seasons; D); and yield–scaled 15N recovery related to the total biomass production in the first (E), and second (F) growing seasons in response to soil amendments [control, phosphogypsum (PG), lime (L), and lime + phosphogypsum (LPG)]. Different lowercase letters indicate significant differences between treatments by Fisher's protected LSD test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) ............................................................................................. 226 Figure 4 – 15N retention at seven stratified soil layers up to 100 cm depth after first (A), and second (B) growing seasons in response to soil amendments [control, phosphogypsum (PG), lime (L), and lime + phosphogypsum (LPG)]. Different lowercase letters for each soil layer indicate significant differences between treatments by Fisher's protected LSD test at p ≤ 0.05. Error bars express the standard error of the mean (n = 4) ...... 228 Figure 5 – Copy numbers of phylogenetic (16S rRNA) marker genes of bacteria (A) and archaea (B), and functional marker genes related to N cycle as amoB (C), amoA (D), nirK (E) and nosZ (F) during maize and soybean seasons in response to soil amendments [control, phosphogypsum (PG), lime (L), and lime + phosphogypsum (LPG)]. Different lowercase letters indicate significant differences between treatments by Fisher's protected LSD test at p ≤ 0.05. Error bars express the standard error of the mean (n = 12) ............................................................................ 229 Figure 6 – Redundancy analysis (RDA) triplot showing the relationship between the soil fertility × 15N recovery from both first and second growing seasons (A), soil fertility × N microbial genes from both first and second growing seasons (B), soil microbial genes × 15N recovery form first (C) and second (D) growing seasons. Canonical axes are labeled with percentage of total variance (RDA1 = main variation; RDA2 = remaining variation). Arrows indicate correlations between variables. The significance of these correlations was assessed by Monte Carlo permutation test with 999 permutations. Significant variables (p ≤ 0.05) are indicated by an asterisk. Color dashed lines indicate significant (p ≤ 0.05) clusters by permutation analysis (PERMANOVA, p ≤ 0.05). First season variables are represented by red color, whereas second season are represented by green color (A and B). SOC = soil organic carbon; 15N rec. = 15N recovery; SDW = stover dry weight; GDW = grain dry wight; RDW = ruzigrass biomass dry weight; 16S rRNA bac. = 16S rRNA bacteria; 16S rRNA arc. = 16S rRNA archaea ..................... 231 Supplementary material Figure S1 – Heatmap of the Pearson’s correlation coefficients among the total abundance of N–cycle functional genes. Only significant correlations (p≤0.05 ) are not marked with “×” ................................................... 245 LISTA DE TABELAS Chapter 1 – Improving soil fertility with lime and phosphogypsum enhances soybean yield and physiological characteristics Supplementary material Table S1 – Crops and treatments application scheme during the experimental period (from 2002 to 2019) ................................................................ 75 Table S2 – Statistical parameters by ANOVA and Student's t-test at p ≤ 0.05 significance level of experimental factors [control (no soil amendment application), lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG)] for soil chemical properties in stratified layers (0.0–1.0 m depth) .............................................................................. 76 Table S3 – Average and statistical parameters for soil organic matter (SOM), phosphorus (P), iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) at 0.0–0.2 m depth affected by different treatments [control, phosphogypsum (PG), lime (L), and lime + phosphogypsum (LPG)] ............................................................................................... 76 Table S4 – Statistical parameters by ANOVA and Student's t-test at p ≤ 0.05 significance level of experimental factors [control (no soil amendment application), lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) × two growing seasons] for soybean root dry matter and root dry matter distribution in stratified layers (0.0–1.0 m depth) ................................................................................................ 77 Table S5 – Influence of lime (L) phosphogypsum (PG), and lime + phosphogypsum (LPG) on nutrient concentration in the leaves of soybean cultivated in two growing seasons in a long-term no-till system ............................ 77 Table S6 – Statistical parameters by ANOVA and Student's t-test at p ≤ 0.05 significance level of experimental factors [control (no soil amendment application), lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) × two growing seasons] for nutritional status of soybean ............................................................................................ 78 Table S7 – Statistical parameters by ANOVA and Student's t-test at p ≤ 0.05 significance level of experimental factors [control (no soil amendment application), lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) × two growing seasons] for soybean physiological, biochemical and agronomic parameters ...................... 79 Chapter 2 – Long-term lime and phosphogypsum amended-soils alleviates the field drought effects on carbon and antioxidative metabolism of maize by improving soil fertility and root growth Table 1 – Influence of surface-applied lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) on nutrient (N, P, K, Ca, Mg, S, Fe, Mn, Cu and Zn) concentrations in the leaves of maize cultivated in two growing seasons in a long-term no-till system ................................................ 97 Supplementary material Table S1 – Crops growing and treatments application scheme during the experimental period (from 2002 to 2018) ........................................ 120 Table S2 – Statistical parameters by ANOVA and Student's t-test at p ≤ 0.05 significance level of experimental factors [control (no soil amendment application), lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG)] for soil chemical properties in stratified layers (0.0–1.0 m depth) ........................................................................... 121 Table S3 – Statistical parameters by ANOVA and Student's t-test at p ≤ 0.05 significance level of experimental factors [control (no soil amendment application), lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG)] for soil chemical properties at 0.0–0.2 m depth .............................................................................................. 121 Table S4 – Statistical parameters by ANOVA and Student's t-test at p ≤ 0.05 significance level of experimental factors [control (no soil amendment application), lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) × two growing seasons] for maize root dry matter and root dry matter distribution in stratified layers (0.0–1.0 m depth) ............................................................................................. 122 Table S5 – Statistical parameters by ANOVA and Student's t-test at p ≤ 0.05 significance level of experimental factors [control (no soil amendment application), lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) × two growing seasons] for nutritional status of maize.............................................................................................. 123 Table S6 – Statistical parameters by ANOVA and Student's t-test at p ≤ 0.05 significance level of experimental factors [control (no soil amendment application), lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG) × two growing seasons] for maize physiological, biochemical, and agronomic parameters ........................................ 124 Chapter 3 – Modulation of the soil microbiome by long-term ca-based soil amendments boosts soil organic carbon and physicochemical quality in a tropical no-till crop rotation system Table 1 – Soil fertility (with available nutrients) and physical parameters of soil long-term amended with phosphogypsum (PG), lime (L) and combined lime and phosphogypsum (LPG) applied in soil surface ........................................................................................... 137 Supplementary material Table S1 – Soil physicochemical properties of the field experimental prior to experiment installation .................................................................... 165 Table S2 – Crop history and the treatment application scheme throughout the experimental period (2002–2019) ................................................... 166 Table S3 – Statistical parameters by ANOVA and Student's t-test at p ≤ 0.05 significance level according to the soil amendments [control (no soil amendment application), lime (L), phosphogypsum (PG), and lime + phosphogypsum (LPG)] for soil organic matter fractions, and soil physicochemical parameters ........................................................... 170 Table S4 – Summary of affected taxa by treatments ......................................... 171 Table S5 – List of bacteria (genus level) significantly affected by treatments .... 172 Table S6 – List of fungi (species level) significantly affected by treatments ....... 174 Chapter 4 – Long-term lime and gypsum amendment increase nitrogen fixation and decrease nitrification and denitrification gene abundances in the rhizosphere and soil in a tropical no-till intercropping system Table 1 – Soil chemical properties .................................................................. 186 Supplementary material Table S1 – Crops growing and treatments application scheme during the experimental period (from 2002 to 2019) ......................................... 207 Table S2 – Description of primers, standards DNA and amplification conditions that were used in qPCR analysis ........................................................... 208 Chapter 5 – Co–application of lime and phosphogypsum increases 15N recovery and reduces its losses by modulating soil fertility, crop growth and n cycle genes Table 1 – Soil fertility in response to soil amendments [control (no soil amendments applied), phosphogypsum (PG), lime (L), and lime + phosphogypsum (LPG)] at seven stratified soil layers up to 100 cm depth ............................................................................................... 224 Supplementary material Table S1 – Crop history and the treatment application scheme throughout the experimental period (2002–2020) .................................................... 243 Table S2 – Description of primers, standards DNA and amplification conditions that were used in qPCR analysis ........................................................... 244 SUMMARY GENERAL INTRODUCTION ..................................................................... 33 CHAPTER 1 - IMPROVING SOIL FERTILITY WITH LIME AND PHOSPHOGYPSUM ENHANCES SOYBEAN YIELD AND PHYSIOLOGICAL CHARACTERISTICS .................................................. 35 1.1 INTRODUCTION ....................................................................................... 36 1. 2 MATERIAL AND METHODS ..................................................................... 38 1.2.1 Site description and experimental design .................................................. 38 1.2.2 Crop sowing and establishment ................................................................. 40 1.2.3 Meteorological data ................................................................................... 40 1.2.4 Soil chemical properties analysis ............................................................... 43 1.2.5 Root dry matter .......................................................................................... 43 1.2.6 Nutrient concentrations in leaves and grains ............................................. 44 1.2.7 Gas exchange parameters ........................................................................ 44 1.2.8 Physiological analysis of plant leaves ........................................................ 45 1.2.8.1 Photosynthetic pigments ........................................................................... 45 1.2.8.2 Rubisco (EC 4.1.1.39), and sucrose synthase (Susy, EC 2.4.1.13) activities .................................................................................................... 45 1.2.8.3 Sucrose concentration ............................................................................... 46 1.2.8.4 Hydrogen peroxide .................................................................................... 47 1.2.8.5 Lipid peroxidation ...................................................................................... 47 1.2.8.6 Total soluble protein .................................................................................. 47 1.2.8.7 Superoxide dismutase (SOD, EC:1.15.1.1) ............................................... 48 1.2.8.8 Catalase (CAT, 1.11.1.6) ........................................................................... 48 1.2.8.9 Ascorbate peroxidase (APX, EC:1.11.1.11) ............................................... 48 1.2.8.10 Glutathione reductase (GR, EC 1.6.4.2) .................................................... 49 1.2.9 Shoot dry matter and grain yield ................................................................ 49 1.2.10 Statistical analysis ..................................................................................... 49 1.3 RESULTS .................................................................................................. 50 1.3.1 Climatic conditions..................................................................................... 50 1.3.2 Soil chemical properties and root development in the soil profile ............... 50 1.3.3 Nutritional status of plants ......................................................................... 53 1.3.4 Photosynthetic metabolism ........................................................................ 54 1.3.5 Oxidative stress and antioxidant metabolism ............................................ 56 1.3.6 Soybean shoot dry matter production, grain yield and crude protein in grains ........................................................................................................ 58 1.3.7 Redundancy analysis and Pearson’s correlation analysis of environmental factors (soil chemical properties) and soybean plant parameters .............. 59 1.4 DISCUSSION ........................................................................................... 61 1.4.1 Weather conditions ................................................................................... 61 1.4.2 Soil chemical properties, root development and nutritional status of plants ........................................................................................................ 61 1.4.3 Leaf pigments, gas exchange and carbon metabolism ............................. 63 1.4.4 Antioxidant metabolism ............................................................................. 65 1.4.5 Soybean shoot dry matter production, grain yield and crude protein in grains ........................................................................................................ 65 1.5 CONCLUSIONS ....................................................................................... 66 REFERENCES ......................................................................................... 68 SUPPLEMENTARY MATERIAL ............................................................... 75 CHAPTER 2 - LONG-TERM LIME AND PHOSPHOGYPSUM AMENDED- SOILS ALLEVIATES THE FIELD DROUGHT EFFECTS ON CARBON AND ANTIOXIDATIVE METABOLISM OF MAIZE BY IMPROVING SOIL FERTILITY AND ROOT GROWTH ........................................................... 81 2.1 INTRODUCTION ...................................................................................... 82 2.2 MATERIAL AND METHODS ..................................................................... 84 2.2.1 Site description, experimental design and treatments ............................... 84 2.2.2 Crop management .................................................................................... 85 2.2.3 Meteorological data .................................................................................. 85 2.2.4 Soil chemical properties analysis .............................................................. 87 2.2.5 Root sampling and dry matter determination ............................................. 87 2.2.6 Leaves sampling for crop nutrition and physiologic analysis ..................... 87 2.2.7 Shoot dry matter and grain yield of maize ................................................. 92 2.2.8 Statistical analysis .................................................................................... 93 2.3 RESULTS ................................................................................................. 93 2.3.1 Weather conditions ................................................................................... 93 2.3.2 Soil fertility and root development ............................................................. 93 2.3.3 Plant nutrition ............................................................................................ 97 2.3.4 Photosynthetic pigments and gas exchange measurements ..................... 97 2.3.5 Carbon metabolism ................................................................................... 99 2.3.6 Lipid peroxidation and antioxidant metabolism ........................................ 100 2.3.7 Maize shoot dry matter production and grain yield ................................... 102 2.3.8 Redundancy and correlation analyses of soil and maize plant measurements ......................................................................................... 103 2.4 DISCUSSION .......................................................................................... 106 2.4.1 Climatic conditions................................................................................... 106 2.4.2 Changes in soil chemical properties, root development and plant nutrition ................................................................................................... 106 2.4.3 Photosynthetic parameters and carbon metabolism response ................. 108 2.4.4 Lipid peroxidation and antioxidant metabolism response ......................... 110 2.4.5 Maize shoot dry matter production and grain yield ................................... 111 2.5 CONCLUSIONS ...................................................................................... 112 REFERENCES ........................................................................................ 113 SUPPLEMENTARY MATERIAL .............................................................. 120 CHAPTER 3 - MODULATION OF THE SOIL MICROBIOME BY LONG- TERM CA-BASED SOIL AMENDMENTS BOOSTS SOIL ORGANIC CARBON AND PHYSICOCHEMICAL QUALITY IN A TROPICAL NO-TILL CROP ROTATION SYSTEM ................................................................... 126 3.1 INTRODUCTION ..................................................................................... 127 3.2 MATERIAL AND METHODS ................................................................... 129 3.2.1 Field description ...................................................................................... 129 3.2.2 Experimental setup, treatments and cropping history .............................. 130 3.2.3 Soil sampling and analysis ...................................................................... 131 3.2.3.1 Soil physicochemical properties............................................................... 131 3.2.3.2 Soil organic matter physicochemical fractionation ................................... 133 3.2.3.3 Soil DNA extraction ................................................................................. 134 3.2.3.4 Amplification, sequencing and sequence processing of 16S rRNA and ITS data ......................................................................................................... 134 3.2.4 Statistical Analyses.................................................................................. 135 3.3 RESULTS ................................................................................................ 136 3.3.1 Soil physicochemical parameters ............................................................ 136 3.3.2 Labile and stable C fractions and aboveground dry matter production by maize and ruzigrass .................................................................................137 3.3.3 Impact of Ca-based soil amendments on the soil microbiome ..................139 3.4 DISCUSSION ..........................................................................................147 3.4.1 Ca-based soil amendments change soil physicochemical attributes and shape soil chemical and physical carbon fractions ...................................147 3.4.2 Linking the soil microbiome with the legacy of Ca-based soil amendments for soil quality ...........................................................................................150 3.5 CONCLUSIONS ......................................................................................154 REFERENCES ........................................................................................155 SUPPLEMENTARY MATERIAL ..............................................................165 CHAPTER 4 - LONG-TERM LIME AND GYPSUM AMENDMENT INCREASE NITROGEN FIXATION AND DECREASE NITRIFICATION AND DENITRIFICATION GENE ABUNDANCES IN THE RHIZOSPHERE AND SOIL IN A TROPICAL NO-TILL INTERCROPPING SYSTEM ........176 4.1 INTRODUCTION .....................................................................................177 4.2 MATERIAL AND METHODS ....................................................................180 4.2.1 Field description and sample collection ....................................................180 4.2.2 Experimental design and treatments ........................................................180 4.2.3 Lime and gypsum application, tillage and crop management ...................182 4.2.4 Soil chemical properties analysis .............................................................183 4.2.5 Soil DNA extraction ..................................................................................183 4.2.6 Quantitative PCR analyses ......................................................................184 4.2.7 Determination of plant nitrogen content and crop yield.............................184 4.2.8 Statistical analysis ...................................................................................185 4.3 RESULTS ................................................................................................185 4.3.1 Soil chemical properties ...........................................................................185 4.3.2 N cycle functional genes (real-time PCR) .................................................186 4.3.3 Redundancy analysis (RDA) and correlation between the relative abundance of N cycle functional genes and soil chemical properties .......190 4.3.4 N content in maize and ruzigrass leaves and maize grain yield ...............192 4.4 DISCUSSION ..........................................................................................193 4.4.1 Changes in soil chemical properties .........................................................193 4.4.2 N cycle genes in an intercropped system with soil amendment ................195 4.4.3 N content in maize and ruzigrass leaves and maize grain yield ...............198 4.5 CONCLUSIONS ...................................................................................... 199 REFERENCES ........................................................................................ 200 SUPPLEMENTARY MATERIAL .............................................................. 207 CHAPTER 5 - CO–APPLICATION OF LIME AND PHOSPHOGYPSUM INCREASES 15N RECOVERY AND REDUCES ITS LOSSES BY MODULATING SOIL FERTILITY, CROP GROWTH AND N CYCLE GENES .................................................................................................... 212 5.1 INTRODUCTION ..................................................................................... 213 5.2 MATERIAL AND METHODS ................................................................... 215 5.2.1 Site description ........................................................................................ 215 5.2.2 Experimental design and field management ............................................ 217 5.2.3 Crop management ................................................................................... 218 5.2.4 15N microplots establishment ................................................................... 218 5.2.5 Sampling procedure of 15N–labeled material and isotopic analyses ......... 219 5.2.6 15N calculations ....................................................................................... 220 5.2.7 Soil sampling and chemical analyses ...................................................... 221 5.2.8 Soil DNA sampling, extraction and qPCR analyses ................................. 221 5.2.9 Statistical analysis ................................................................................... 222 5.3 RESULTS ................................................................................................ 222 5.3.1 Soil profile fertility .................................................................................... 222 5.3.2 Aboveground dry matter yield .................................................................. 224 5.3.3 15N Recovery in soil–plant system ........................................................... 225 5.3.4 15N retention along the soil profile ............................................................ 228 5.3.5 Phylogenetic and functional (N cycle) marker genes ............................... 228 5.3.6 Linking plant–soil–genes data ................................................................. 230 5.4 DISCUSSION .......................................................................................... 232 5.4.1 Soil profile fertility .................................................................................... 232 5.4.2 Aboveground dry matter yield and fate of 15N–labeled fertilizer ............... 233 5.4.3 Distribution of 15N–fertilizer along soil profile and soil microbial genes..... 235 5.5 CONCLUSIONS ...................................................................................... 237 REFERENCES ........................................................................................ 237 SUPPLEMENTARY MATERIAL .............................................................. 243 GENERAL CONCLUSIONS .................................................................... 246 REFERENCES ........................................................................................ 249 33 GENERAL INTRODUCTION Soil acidity is one of the most limiting factors for crop development by reducing its productive potential. Approximately 50% of the arable area across the globe is affected by problems related to soil acidity (SUMNER; NOBLE, 2003; VON UEXKÜLL; MUTERT, 1995). Most of these soils are located in tropical and subtropical regions, as they present a high degree of weathering, which promotes acidity problems, reduced cation exchange capacity (CEC) and base saturation (BS), in addition to containing high levels of toxic elements such as exchangeable aluminum (Al3+) (CAIRES et al., 2008; CAIRES et al., 2011; CRUSCIOL et al., 2016; FAGERIA; NASCENTE, 2014; LI et al., 2019). Brazil, one of the largest food producers in the world, has about 58% of its territory naturally acidic, which becomes a major problem especially in low-altitude Cerrado, a Biome with predisposition to dry winters and summers with dry spell periods (CUNNINGHAM, 2020). Liming is a very important practice for the viability of agricultural production, since this operation can neutralize soil acidity, increasing nutrient availability, in addition to providing calcium (Ca2+) and magnesium (Mg2+), and reducing Al3+ toxicity, especially at uppermost soil layers. On the other hand, the no-tillage system (NTS) requires no tilling of the soil, reducing liming efficiency (CAIRES; GARBUIO; BARTH, 2008; SANTOS et al., 2018). In this type of production system, liming is usually carried out on the soil surface, without incorporation, promoting great inquiries in the scientific environment due to low low mobility in the profile (low solubility in water) (SORATTO; CRUCIOL, 2008). Due to the issues that occurs in surface application of lime, studies with phosphogypsum has started. Phosphogypsum is a by-product of obtaining phosphoric acid, basically composed of calcium sulfate (CaSO4.2H2O). This input, unlike lime, does not correct soil acidity; however it is an alternative in providing Ca2+ at deep layers, in addition to reducing Al3+ toxic levels throughout the soil profile (CARMEIS FILHO; CRUSCIOL; CASTILHOS, 2017; CRUSCIOL et al., 2019; SORATTO; CRUSCIOL, 2008). Phosphogypsum solubility is higher compared with lime (~150 times), and thus, moving more quickly through the soil profile (COSTA et al., 2018; TIRITAN et al., 2016). Phosphogypsum, therefore, has been used as a complementary input to lime, enhancing the liming benefits to the soil (BOSSOLANI et al., 2020; CRUSCIOL et al., 2019). On the other hand, there are many formulas for 34 recommending phosphogypsum for Brazilian soils (CAIRES; GUIMARÃES, 2018; VAN RAIJ et al., 2001; VITTI; LUZ, 2004), requiring further investigation as to the best method for each type of agricultural system and soil. In addition to the benefits for soil chemical attributes, the application of lime, combined or not with phosphogypsum, can also improve soil physics (soil structure), although this effect is not yet fully understood (CARMEIS FILHO et al., 2017; ROTH; PAVAN, 1991). Regarding microbiological indicators of soil quality, the effect of lime and phosphogypsum management has not yet been elucidated, and contradictory results are often obtained, mainly due to the spatial heterogeneity and complexity of the system (BADALUCCO et al., 1992). These results become more incipient when changes occur only over long-term periods (CUSSER et al., 2020; DUVAL et al., 2013). Liming can increase soil microbial activity (BOSSOLANI et al., 2020; HOLLAND et al., 2018). As a rule, raising the soil pH, increases the mineralization of soil organic matter, in addition to providing a direct impact on the nitrogen cycle potential of soils (BOSSOLANI et al., 2020; HOLLAND et al., 2018). Studies in tropical regions evaluating the effect of lime and phosphogypsum on the soil microbiological activity and structure are scarce, and further investigations are necessary, given the importance of microbiology in increasing the productivity of soil and crops (BOSSOLANI et al., 2021a). Therefore, understanding the effect of these inputs on soil quality is important to predict changes in soil productive balance and the long-term sustainability, and profitability of agricultural systems (GROVER et al., 2017; INAGAKI et al., 2016), especially in tropical region. Furthermore, soil fertility management with lime and phosphogypsum can be used as an important tool to overcome periods of water scarcity (BOSSOLANI et al., 2021b). Tropical regions presents stronlgy predisposition to dry periods throughout the year CARMEIS FILHO et al., 2017, which drastically reduce crop yield. The construction of a fertile profile ensures increased root growth of crops, accessing a greater amount of soil resources (water and nutrients), which leads crops to overcome periods of drought (BOSSOLANI et al., 2021b) Therefore, this research aimed to evaluate the effect of surface application of lime and phosphogypsum on the combination of soil chemical with physical and biological properties (functional genes and taxonomical profile), root growth, plant nutrition, carbon and antioxidant metabolism, yield components, and the 15N-fertilizer recovery by crops in the soil-plant system. 246 GENERAL CONCLUSIONS Our results advances the understanding of the dynamics of surface lime application and the combined effect of lime and phosphogypsum in the different aspects that make up a long-term tropical no-till system. Our results showed that the application of lime alone and especially in combination with phosphogypsum improves soil fertility up to 1 m deep, culminating in increased root growth of soybean and maize crops. As a result, both crops increased the leaf nutrient concentrations, reflecting an improvement in the concentration of leaf photosynthetic pigments, and gas exchange (net photosynthetic rate, stomatal conductance, substomatal concentration of CO2, leaf transpiration and water use efficiency), and on the activities of Rubisco and SuSy enzymes. Interestingly, when studying the water balance during the soybean and maize growing cycle, we confirmed periods of water scarcity, and when lime and phosphogypsum were applied, drought stress (measured through antioxidant enzymes and lipid peroxidation) reduced. Crop grain yield was also increased with liming, and even more expressive results occurred by the combination of lime and phosphogypsum. Interestingly, soil microbiology highly contributed to the understanding of the dynamics of soil-plant relationship of this study. The productive responses of soybean and maize did not occur only due to changes in the soil chemical properties. Deepening the understanding of tropical agricultural systems altered by the application of lime and phosphogypsum, our results revealed that the joint application of these inputs changed the soil chemical properties in tropical intercropping system managed under no-till, increasing the relative abundance of total prokaryotes (archaea and bacteria), modulating genes related to the nitrogen cycle. Exchangeable Ca2+ and Mg2+ were the main nutrients related to the increase of genes related to nitrogen fixation and to the reduction of the relative abundance of genes related to nitrification and denitrification (even though the total abundance increased), for both soil and rhizospheres of maize and ruzigrass. In addition, the synergism between lime and phosphogypsum also increased the quantity and quality of soil organic matter, mainly by altering the relationship between bacteria and fungi communities of the soil. Interestingly, we found that soil improvers act by modifying soil quality at a hierarchical level as follows: soil fertility > microbiome (fungi + bacteria) > chemical fractions of soil organic matter > soil physics > physical fractions of soil organic matter. Finally, our results also revealed that the combined application of these inputs has a strong 247 influence on the selection of microorganisms that are not abundant in the soil (rare community), reflecting a potential increase in the multifunctionality of the soil. Our results also showed that the increase in the abundance of related genes in soils managed with lime and phosphogypsum favors the recovery of 15N fertilizer, which strongly reduces the losses of nitrogen fertilizer. On the other hand, we saw that the isolated use of phosphogypsum has a potential to leaching N to deep layers. In the absence of the management with lime and phosphogypsum (control treatment), the abundance of genes related to denitrification has increased, which indicates a high potential for denitrification in these soils, increasing even more the N losses in the system. 248 249 REFERENCES BADALUCCO, L.; GREGO, S.; DELL’ORCO, S.; NANNIPIERI, P. Effect of liming on some chemical, biochemical, and microbiological properties of acid soils under spruce (Picea abies L.). Biology and Fertility of Soils, v. 14, p. 76-83, 1992. BOSSOLANI, J. W.; CRUSCIOL, C. A. C.; LEITE, M. F. A.; MERLOTI, L. F.; MORETTI, L. G.; PASCOALOTO, I. M.; KURAMAE, E. E. Modulation of the soil microbiome by long-term Ca-based soil amendments boosts soil organic carbon and physicochemical quality in a tropical no-till crop rotation system. Soil Biology and Biochemistry, v. 156, 108188, 2021a. DOI: https://doi.org/10.1016/j.soilbio.2021.108188 BOSSOLANI, J. W.; CRUSCIOL, C. A. C.; GARCIA, A.; MORETTI, L. G.; PORTUGAL, J. R.; RODRIGUES, V. A.; FONSECA, M. C.; CALONEGO, J. C.; CAIRES, E. F.; AMADO, T. J. C.; REIS, A. R. D. Long-term lime and phosphogypsum amended-soils alleviates the field drought effects on carbon and antioxidative metabolism of maize by improving soil fertility and root growth. Frontiers in Plant Science, 1437, 2021b. https://doi.org/ 10.3389/fpls.2021.650296. BOSSOLANI, J. W.; CRUSCIOL, C. A. C.; MERLOTI, L. F.; MORETTI, L. G.; COSTA, N. R.; TSAI, S. M.; KURAMAE, E. E. Long-term lime and gypsum amendment increase nitrogen fixation and decrease nitrification and denitrification gene abundances in the rhizosphere and soil in a tropical no-till intercropping system. Geoderma, n. 375, 114476, 2020. DOI: https://doi.org/10.1016/j.geoderma.2020.114476 CAIRES, E. F.; GARBUIO, F. J.; BARTH, G. 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