RESSALVA 
 

 

 

Atendendo solicitação do(a) autor(a), o 

texto completo desta tese/dissertação será 

disponibilizado somente a partir de 

29/01/2022. 



 

 

 

 

 CAMILA DA SILVA GRASSMANN 

 

 

 

 

 

MAIZE-BASED SYSTEMS AS AFFECTED BY FORAGE GRASS AND NITROGEN 

FERTILIZATION: ELUCIDATING 15N-FERTILIZER RECOVERY, GREENHOUSE 

GAS EMISSIONS, N-CYCLE FUNCTIONAL GENES IN SOIL AND CROP YIELDS 

 

 

 

 

 

 

 

 

 

 

Botucatu 

2021 



 



 
 

CAMILA DA SILVA GRASSMANN 

 

 

 

 

 

 

 

 

 

 

MAIZE-BASED SYSTEMS AS AFFECTED BY FORAGE GRASS AND NITROGEN 

FERTILIZATION: ELUCIDATING 15N-FERTILIZER RECOVERY, GREENHOUSE 

GAS EMISSIONS, N-CYCLE FUNCTIONAL GENES IN SOIL AND CROP YIELDS 

 

 

 

 

Doctoral thesis submitted to the College of 

Agricultural Sciences, UNESP - Botucatu 

Campus, to obtain the degree of Doctor in 

Agriculture. 

 

Advisor: Prof. Dr. Ciro Antonio Rosolem 

Co-advisor: Dr. Eduardo Mariano 

 

 

Botucatu 

2021  



G769m
Grassmann, Camila da Silva

    Maize-based systems as affected by forage grass and nitrogen fertilization:

Elucidating 15N recovery, greenhouse gas emissions, N-cycle functional genes

in soil and crop yields / Camila da Silva Grassmann. -- Botucatu, 2021

    159 p. : il., tabs.

    Tese (doutorado) - Universidade Estadual Paulista (Unesp), Faculdade de

Ciências Agronômicas, Botucatu

    Orientador: Ciro Antonio Rosolem

    Coorientador: Eduardo Mariano

    1. Soil fertility. 2. Nitrogen cycle. 3. Forage plants Soils. 4. Greenhouse

gases. I. Título.

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TÍTULO DA TESE:  

UNIVERSIDADE ESTADUAL PAULISTA  

 

Câmpus de Botucatu  

 

 
CERTIFICADO DE APROVAÇÃO  

 

 

 
MAIZE-BASED SYSTEMS AS AFFECTED BY FORAGE GRASS AND NITROGEN 
FERTILIZATION: ELUCIDATING 15N-FERTILIZER RECOVERY, GREENHOUSE 
GAS EMISSIONS, N-CYCLE FUNCTIONAL GENES IN SOIL AND CROP YIELDS 

 

 

AUTORA: CAMILA DA SILVA GRASSMANN 

ORIENTADOR: CIRO ANTONIO ROSOLEM 

COORIENTADOR: EDUARDO MARIANO  

 

 

 

Aprovada como parte das exigências para obtenção do Título de Doutora em AGRONOMIA 

(AGRICULTURA), pela Comissão Examinadora:  

 

 

 
Prof. Dr. CIRO ANTONIO ROSOLEM (participação Virtual)  
Produção Vegetal  / Faculdade de Ciências Agronômicas de Botucatu - UNESP  

 

Pesquisador Dr. HEITOR CANTARELLA (participação Virtual)  
Centro de Pesquisa e Desenvolvimento de Solos e Recursos Ambientais / Instituto Agronômico de Campinas  

 

Prof. Dr. RAFAEL OTTO (participação Virtual)  
Ciência do Solo / Escola Superior de Agricultura Luiz de Queiroz  

 

Prof. Dr. PAULO SÉRGIO PAVINATO (participação Virtual)  
Departamento de Ciência do Solo / ESCOLA SUPERIOR DE AGRICULTURA  

 

PROF. DR. GUSTAVO CASTOLDI (Participação Virtual), 

POLO DE INOVAÇÃO / INSTITUTO FEDERAL GOIANO  

 

 

Botucatu, 29 de janeiro de 2021  

 

 

 

 

 

 

 

 

 
Faculdade de Ciências Agronômicas - Câmpus de Botucatu - 

Avenida Universitária, , 3780, 18610034, Botucatu - São Paulo  



 
 

  



 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I dedicate my dissertation work to my family and friends. A special feeling of gratitude 

to my loving parents Carlos and Sandra Grassmann whose words of encouragement 
and unconditional love was essential. My special brother Guilherme, who never left 

my side. 
 

I also dedicate this dissertation to my boyfriend, for supporting me, understanding 

and loving me. 



 
 

 



 
 

ACKNOWLEDGEMENTS 

Firstly, and always to God, for health and strength to work, and enlightenment to 

discern between right and wrong. 

Prof. Dr. Ciro Antonio Rosolem, for his friendship, advice, teaching, trust, partnership 

and excellent support in these almost 8 years of orientation that started in the 

undergrad. I will be forever grateful for the opportunity granted. 

Prof. Dr. Karl Ritz, from the University of Nottingham, for the excellent guidance and 

teaching, as well as friendship. 

Dr. Eduardo Mariano, who I feel honored and grateful to have had as a co-supervisor. 

I will be forever grateful for so much learning and friendship. 

Prof. Dr. Paulo Roberto Miranda Meirelles and Dr. André Michel de Castilhos, for the 

support in analysis involving zootechnical matters. 

Bruno Rosolen Gilli, for all the help, encouragement and companionship in field 

sampling and laboratory determinations. 

São Paulo State University (UNESP), College of Agricultural Sciences, for the 

teaching, infrastructure and support offered. 

Graduate Program in Agriculture - UNESP/FCA, for the PhD opportunity. 

Professors and employees of the Graduate Program in Agronomy (Agriculture) of the 

São Paulo State University “Júlio de Mesquita Filho” (Botucatu Campus), for the 

teaching and collaboration. 

Employees of the Plant Production Department, especially Adelina, Casemiro, Dariele, 

Vinícius, Ciro, Dorival, Eliane, Iara, Júlia, Mateus, Valéria, and Vinícius for their 

friendship, collaboration and teaching. 

All members of NUCLEUS: A virtual joint centre to deliver enhanced N–use efficiency 

via an integrated soil–plant systems approach for the United Kingdom and Brazil. 

Funded in Brazil by FAPESP–São Paulo Research Foundation [grant number 

2015/50305–8]. 



 
 

This work was carried out with the support of the São Paulo State Research Support 

Foundation (FAPESP), for granting a PhD scholarship (grant number 2016/25253-7) 

and BEPE scholarship (grant number 2018/09622-8) and Coordination for the 

Improvement of Higher Education Personnel-Brazil (CAPES) – Financing code 001. 

To the friends of the Graduate Program, especially Eduardo Mariano, Carlos 

Nascimento, Bruno Gazola, Sérgio Freitas, Gustavo Ferreira, Laudelino Motta, 

Barbara Silva, Priscila Bahia, Beatriz Borges, and Clóvis Borges, for the moments of 

partnership, friendship, collaboration and support. 

To the interns Bruno Rosolen Gilli, Rodrigo Martins, Lucas Brunini, Thiago Benetom, 

Fernando Thome, and Matheus Miziara for their friendship and help in carrying out this 

work. 

Employees and teachers of UNASP/SP, who contributed to my personal and school 

education. 

All those who, directly or indirectly, contributed to the development realization of this 

study, my sincere and eternal thanks. 

THANKS! 

 

 

 

 

 

 

 

 

 

 

 



 
 

ABSTRACT 

Due to the interest in N use efficiency (NUE) and sustainable agricultural systems, the 
adoption of integrated systems, such as the intercropping of maize with forage grasses 
can be of great relevance, allowing the use of the land throughout the year, besides 
avoiding losses of N through nitrate (NO3

-) leaching, nitrous oxide (N2O) emissions, 
ammonia volatilization (NH3), and immobilization. Tropical forage grasses of the genus 
Megathyrsus and Urochloa can suppress soil–nitrification by releasing inhibitory 
substances, reducing N losses and increasing fertilizer N recovery of the cash crop in 
rotation. In this way, understanding the N transformations in the soil by microorganisms 
and the fertilizer recovery in the system are very important. Firstly, the first two chapters 
are about a 3-year (2014-2017) field experiment conducted in southeastern Brazil, 
were forage grasses Guinea grass (Megathyrsus maximus cv. Tanzânia), palisade 
grass (Urochloa brizantha cv. Marandu), and ruzigrass (Urochloa ruziziensis cv. 
Comum) were cultivated in rotation with maize for grain  in summer, to analyze the 
influence of forage grass and N fertilization in each study. In first chapter, maize was 
fertilized with 140 kg ha-1 N as (15NH4)2SO4 or not fertilized, and recovery of residual 
15N was quantified in the second season. In second chapter, the change was that the 
N source used was ammonium sulphate not labeled, and were analyzed nitrous oxide 
(N2O), methane (CH4), and NH3 emissions from the system. In the third and fourth 
chapter, maize was intercropped with the same grasses previously mentioned. The N 
rates were 90, 180 and 270 kg ha-1 N and treatments without N fertilization. The 
objective was also to ascertain the effect of grasses and N fertilization from the 
analyzes carried out. The third chapter characterized the changes in N-cycle genes in 
the soil and measured the N2O emissions. The fourth chapter assessed maize grain 
yield and forage production, bromatological quality, and estimated meat production. In 
the first season after 15N application, 21%, 65%, and 33% of the N in maize grain, 
stover, and shoots, respectively, was derived from fertilizer. In the next season, of the 
total N found in maize grain, stover, and shoots, 2.2%, 1.9%, and 2.0%, respectively, 
was derived from the residual fertilizer applied in the previous year. There were no 
differences between forage grass species in the amount of 15N recovered by maize, 
soil, and total N. In the first season of maize in rotation with forage grasses, Guinea 
grass, palisade grass, ruzigrass did not affect N2O and NH3 emission due to their 
apparent inability to suppress soil nitrification. However, N fertilization slightly 
increases cumulative N2O emission. In maize intercropping with grasses, N fertilization 
increases the abundance of AOB (amoA of bacteria) more than AOA (amoA of 
archaea). N2O emission was influenced by AOB, water-filled pore space (WFPS) and 
N fertilization. Nitrogen fertilization positively affects forage growth and nutritional 
quality, resulting in a higher maize grain yield, higher forage production and quality, 
and eventually higher estimated meat production. Moreover, Guinea grass resulted in 
the highest estimated meat production when fertilized with 270 kg ha-1 N. However, no 
evidence of biological inhibition by the grasses were confirmed. 

Keywords: Zea mays L. Urochloa. Megathyrsus. N2O. AOB. AOA. Estimated meat 

production. 

 

 

 



 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 
 

RESUMO 

Devido ao interesse na eficiência no uso do N (NUE) e em sistemas agrícolas 
sustentáveis, a adoção de sistemas integrados, como o consórcio de milho com 
gramíneas forrageiras, pode ser de grande relevância, permitindo o uso da terra ao 
longo do ano, além de evitar perdas de N por lixiviação de nitrato (NO3

-), emissões de 
óxido nitroso (N2O), volatilização de amônia (NH3) e imobilização. Gramíneas 
forrageiras tropicais do gênero Megathyrsus e Urochloa podem suprimir a nitrificação 
do solo ao liberar substâncias inibidoras, reduzindo as perdas de N e aumentando a 
recuperação de N fertilizante da cultura comercial em sucessão. Desta forma, o 
entendimento das transformações do N no solo por microrganismos e a recuperação 
do fertilizante no sistema são muito importantes. Em primeiro lugar, os dois primeiros 
capítulos são a respeito de um experimento de campo de 3 anos (2014-2017) 
conduzido no sudeste do Brasil, onde gramíneas forrageiras capim colonião 
(Megathyrsus maximus cv. Tanzânia), capim braquiária (Urochloa brizantha cv. 
Marandu) e capim braquiária (Urochloa ruziziensis cv. Comum) foram cultivadas em 
rotação com milho para grão no verão, para analisar a influência da gramínea 
forrageira e da fertilização com N em cada estudo. No primeiro capítulo, o milho foi 
fertilizado com 140 kg ha-1 de N na forma de (15NH4)2SO4 ou não fertilizado, e a 
recuperação do 15N residual foi quantificada na segunda safra. No segundo capítulo, 
a mudança foi que a fonte de N utilizada foi o sulfato de amônio não rotulado, e foram 
analisadas as emissões de óxido nitroso (N2O), metano (CH4) e NH3 do sistema. No 
terceiro e quarto capítulos, o milho foi consorciado com as mesmas gramíneas 
mencionadas anteriormente. As doses de N foram 90, 180 e 270 kg ha-1 e os 
tratamentos sem adubação nitrogenada. O objetivo também foi verificar o efeito das 
gramíneas e da fertilização com N a partir das análises realizadas. O terceiro capítulo 
caracterizou as mudanças nos genes do ciclo N no solo e mediu as emissões de N2O. 
O quarto capítulo avaliou o rendimento de grãos de milho e a produção de forragem, 
a qualidade bromatológica e a estimativa da produção de carne. Na primeira safra 
após a aplicação de 15N, 21%, 65% e 33% do N no grão de milho, palha e brotos, 
respectivamente, foi derivado de fertilizante. Na safra seguinte, do total de N 
encontrado nos grãos, caules e ramos de milho, 2,2%, 1,9% e 2,0%, respectivamente, 
foram derivados do fertilizante residual aplicado no ano anterior. Não houve diferenças 
entre as espécies de gramíneas forrageiras na quantidade de 15N recuperado pelo 
milho, solo e N. total. Na primeira temporada de milho em rotação com gramíneas 
forrageiras, capim-Guiné, capim-paliçada, ruzigrass não afetou a emissão de N2O e 
NH3 devido à sua aparente incapacidade de suprimir a nitrificação do solo. No entanto, 
a fertilização com N aumenta ligeiramente a emissão cumulativa de N2O. No consórcio 
de milho com gramíneas, a fertilização com N aumenta a abundância de AOB (amoA 
de bactérias) mais do que AOA (amoA de arquéias). A emissão de N2O foi influenciada 
por AOB, espaço poroso cheio de água (WFPS) e fertilização de N. A fertilização 
nitrogenada afeta positivamente o crescimento das forragens e a qualidade 
nutricional, resultando em maior rendimento de grãos de milho, maior produção e 
qualidade das forragens, e eventualmente maior produção de carne estimada. Além 
disso, o capim colonião resultou na maior produção de carne estimada quando 
fertilizado com 270 kg ha-1 N. No entanto, não foram confirmadas provas de inibição 
biológica por parte das gramíneas. 

Palavras-chave: Zea mays L. Urochloa. Megathyrsus. N2O. AOB. AOA. Produção 

estimada de carne.  



 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 
 

FIGURE LIST 

Figure 1 -  Monthly precipitation, and average minimum and maximum air temperatures   

in the first (2015–2016) and second (2016–2017) growing seasons, and 

long-term average (period 1955-2015)………………………………………32 

Figure 2 -Schematic representation of the whole plot and its microplot (deployed 

exclusively in N–fertilized treatments), in addition to the sampling 

procedures of plant, litter, and soil…………………………………………….36 

Figure 3 - Maize (partitioned into grains and stover) and litter N derived from fertilizer 

as affected by forage grass in the first (2015–2016) and second (2016-2017 

growing season after application of 15N-labeled fertilizer………………….47 

Figure 4 - Soil N derived from fertilizer at four depth layers as affected by forage grass 

in the first (2015–2016) and second (2016–2017) growing season after 

application of 15N-labeled fertilizer…………………………….……………..48 

Figure 5 -The fate of 15N–labeled fertilizer in plant–litter–soil system as affected by 

forage grass in the first (2015–2016) and second (2016–2017) growing 

season after application of 15N-labeled fertilizer……………………………49 

Figure 6 - Daily minimum and maximum air temperatures and rainfall recorded during 

the field experiment period (2014-2017)……………………………………..69 

Figure 7 - Seasonal variation of water-filled pore space (a) and soil temperature (b) at 

depth of 5.5 cm in forage grass-maize rotations from 2015 to 2017 as 

affected by forage grass specie and N fertilization………………………….70 

Figure 8 - Seasonal variation of NH4
+-N (a) and NO3

--N content in the soil (b) at depth 

of 0-10 cm in forage grass-maize rotations from 2014 to 2017 as affected 

by forage grass specie and N fertilization…………………………………….71 

Figure 9 - Seasonal variation of N2O (a) and CH4 flux (b), in addition to NH3 loss rate 

(c) in forage grass-maize rotations from 2014 to 2017 as affected by forage 

grass specie and N fertilization………………………………………………..73 

Supplementary Figure 1 - Cumulative emissions (or uptake) of N2O (a), CH4 (b), and 

NH3 (c) in forage grass-maize rotations as affected by forage grass specie 

and N fertilization over the experimental period…………………………….76 

Supplementary Figure 2 - Cumulative crop biomass (forage plus maize) production (a) 

and yield-scaled emission (or uptake) of N2O (b), CH4 (c), and NH3 (d) in 



 
 

forage grass-maize rotations as affected by forage grass specie and N 

fertilization over the experimental period…………………………………….79 

Figure 10 - Heatmap showing Spearman rank-order correlation coefficients (r) between 

greenhouse gas flux and soil properties (n = 237) throughout the 

experimental period (2015-2017)……………………………………………..80 

Figure 11 - Crop succession occurred in the experimental site from 2014 to 2018….96 

Figure 12- Daily minimum, maximum and average air temperatures and rainfall 

recorded during the growing season period (Dec.2017-May 2018)…….103 

Figure 13 -Contents of NO3
--N (a), NH4

+-N (b), and total N (c) at the 0-20 cm soil layer 

as affected by forage grass specie, N fertilization, and time after 

planting……………………………………………………………………..….105 

Figure 14 - Abundance of 16S rRNA of bacteria (a) and archaea (b and c), nifH (d), 

amoA of bacteria (e), amoA of archaea (f), nirS (g) and nosZ (h) at the 0-20 

cm soil layer as affected by forage grass species, N fertilization and days 

after planting............................................................................................107 

Figure 15 - Cumulative N2O emission in forage grass-maize intercropping as affected 

by forage specie and N fertilization………………………………………….109 

Supplementary Figure 3 - Variation of water-filled pore space (a) and soil temperature 

at depth of 5.5 cm (b) as well as N2O flux (c) in forage grass-maize 

intercropping as affected by forage grass specie and N fertilization…….110 

Figure 16 - Heatmap of the Spearman rank correlation order among the relative 

abundance of N-cycle functional genes, soil properties, and N2O flux. *, **, 

and *** significant at P≤0.05, P≤0.01, and P≤0.001, respectively……….111 

Figure 17 - Principal component analysis of the soil microbial genes (a) and chemical 

properties (b) with N2O fluxes, water-filled pore space (WFPS) and 

temperature (TºC) from soil of 72 sampling points across three 

times………………………..………………………………………….....……113 

Figure 18 - Monthly minimum, maximum and average air temperatures and rainfall 

recorded during the growing season period (Dec.2017-Nov.2018)……..135 

Figure 19 - Forage dry matter yield (a) and maize grain yield (b) at maize harvest. The 

error bars represent the SEM (n = 8 and 4 for panels a and b, 

respectively)…………………………………………………………………..138 



 
 

Figure 20 - Light interception in Guinea grass (a) and palisade grass (b) from 14 days 

after maize harvest until the forage grass reach 95% of LI; SPAD index in 

forage grass cut (c)…………………………………………………………...141 

Figure 21 - Estimated meat production by the LRNS Cornell model…………….....…143 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 
 

TABLE LIST 

Table 1 - Net nitrification rate from 10–d incubation of soil samples taken at the 0–20 

cm depth layer before maize planting in the first (2015–2016) and second 

(2016–2017) growing season, in which the latter was also affected by fertilizer 

N rate………………………………………………………………………..…....39 

Table 2 - Dry matter yield and N accumulation of forage grass species grown before 

maize in the first (2015–2016) and second (2016–2017) growing season, in 

wich the later was also affected by fertilizer N rate…………………………..40 

Table 3 -Maize (partitioned into grains and stover) and litter dry matter yield and 

harvest index (HI) as affected by forage grass and fertilizer N rate in the first 

(2015-2016) and second (2016-2017) growing season……………………..42 

Table 4 – Maize (partitioned into grains and stover) and litter N accumulation and N 

harvest index (NHI) as affected by forage grass and fertilizer N rate in the first 

(2015-2016) and second (2016-2017) growing season……………………..44 

Table 5 - Seasonal N2O and CH4, and NH3 emissions (or uptake) in forage grass-maize 

rotations from 2014 to 2017 as affected by forage grass specie and N 

fertilization………………………………………………………………………..74 

Table 6 - Seasonal crop biomass and yield-scaled (YS) emission (or uptake) of N2O, 

CH4, and NH3in forage grass-maize rotations from 2014 to 2017 as affected 

by forage grass specie and N fertilization ……………………………………77 

Table 7 - Seasonal N2O emission factor in forage grass-maize rotations as influenced 

by forage grasses………………………………………………………………..80 

Table 8 - Description of primers, standards DNA and amplification conditions used in 

qPCR analysis……………………………………………………………………98 

Supplementary Table 1 - Soil properties as affected by N fertilization and days after 

planting (DAP)………………………………………………………………….104 

Supplementary Table 2 - Probability (P) values for microbial genes in the soil as 

affected by forage grass species, N fertilization and days after planting 

(DAP)...………………………………………………………………………….108 

Supplementary Table 3 - Scores of principal components analysis relative to 

temperature (TºC), water-filled pore space (WFPS), N2O flux, and abundance 

of microbial genes as presented in Fig. 17a…………………………………114 

 



 
 

Supplementary Table 4 - Scores of principal components analysis of the soil chemical 

characteristics, water-filled pore space (WFPS), temperature (TºC), and N2O 

flux presented in Fig. 17b………………………………………………………115 

Table 9 - Bromatological quality of forage grass at maize harvest. The error bars 

represent the SEM (n = 16, 8 and 4 for forage grasses, N rate and forage 

grass x N rate, respectively)……………………………………….………..…139 

Table 10 - Dry matter yield and bromatological quality of forage grass at cutting in Nov. 

2018………………………………………………….……………………….....142 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 
 

SUMMARY 

             GENERAL INTRODUCTION……………………………………………………...25 

CHAPTER 1: FATE OF 15N FERTILIZER APPLIED TO MAIZE IN ROTATION 

WITH TROPICAL FORAGE GRASSES…….....………………………………...29 

1.1      Introduction……………………………………………………………………….…30 

1.2      Material and methods………….……………………………….………………….31 

1.2.1   Study site……………………………………………..……………………….…….31 

1.2.2   Study design………….……………..……………………………..……………..…33 

1.2.3   Crop management………………………….……………………..………............33 

1.2.4   Soil nitrification assessed by laboratory incubation………………………….....34 

1.2.5   15N microplots…………………………………………..………………..………….34 

1.2.6   Sampling procedure and 15N analyses …….……………….………………….…36 

1.2.7   Calculations and statistical analysis………………….…………………….…….37 

1.3      Results…………...………………………………………………………………….38 

1.3.1   Net nitrification rates……………………………………………..…………………38 

1.3.2   Forage yield and N accumulation………………………………..……………..…39 

1.3.3   Maize yield and N accumulation………………………………..……………...….40 

1.3.4   Distribution and fate of 15N–labeled fertilizer applied to maize…………………46 

1.4       Discussion……..…………………………………………………………………....49 

1.4.1    Dry matter yield and n accumulation…………………….………………..……..49 

1.4.2    Distribution of 15N–Labeled fertilizer………….…………………………….…….51 

1.4.3    Fate of 15N–labeled fertilizer…………………………….….…………….…..…..52 

1.5      Conclusion…………………………………………..………...…...……….……….55 



 
 

           References……………………………………………………………………….....56 

           CHAPTER 2: EFFECT OF TROPICAL GRASS NITROGEN FERTILIZATION 

ON  NITROUS OXIDE, METHANE, AND AMMONIA EMISSIONS OF MAIZE-

BASED-ROTATION SYSTEMS…………………………………………….…….61 

2.1     Introduction……………………………………….………………………....…...….62 

2.2     Material and methods…………………………………………...…...……….…….64 

2.2.1. Study site and experimental setup……………………….……………………...…64 

2.2.2  Gas sampling procedure and auxiliary measurement……………………..….…65 

2.2.3  Ammonia measurements………………………………………….………………..66 

2.2.4. Calculations and statistical analysis………………………………….……………67 

2.3     Results……………...……………………………………….……………….……….68 

2.3.1  Environmental conditions and soil properties…………………………………….68 

2.3.2  Nitrous oxide, methane, and ammonia emissions………………..……….……...71 

2.3.3  Crop biomass, yield-scaled emissions, and N2O emission factor………………77 

2.3.4  Relationships between greenhouse gas flux and soil properties……………….80 

2.4     Discussion………………..…………………………………………………………..81 

2.4.1  Greenhouse gas flux and rate of ammonia volatilization………………………..81 

2.4.2  Cumulative emissions of greenhouse gases and ammonia…………………….82 

2.4.3   N2O emission factor……………….……………………………………………......84 

2.5       Conclusion…..………………………………………….………………….……….84 

References…………………………………….………..…………..………...…….85 

CHAPTER 3: FUNCTIONAL N-CYCLE GENES IN SOIL AND N2O 

EMISSIONS IN A MAIZE/TROPICAL FORAGE GRASSES INTERCROPPING 

SYSTEM……….……………………………..………………………………….….91 



 
 

3.1       Introduction………………………...…………………………………..…….…..….92 

3.2      Material and methods………….…………………………………...…………....…94 

3.2.1   Site description and experimental design……………..….………………………94 

3.2.2   Soil sampling…………………………………………………..…………………….95 

3.2.3   Soil chemical analysis………………….…………………………………………...96 

3.2.4   Isolation of dna and quantitative real-time PCR…………………………............96 

3.2.5   Gas sampling, temperature and water-filled pore space procedure…………..101 

3.2.6   Statistical analysis……………..……………………………………………..……102 

3.3.     Results……………………………………..……………………………..………..102 

3.3.1  Environmental conditions and soil characteristics………………………………102 

3.3.2  N-cycle functional genes………...……………………………………..………....106 

3.3.3  Nitrous oxide emissions…………………………………………………………...108 

3.3.4  Correlations between the abundance of N cycle genes, soil properties and N2O 

flux…….………………………………………………………………………….…111 

3.3.5   Principal component analysis…………………………………………...……….112 

3.4     Discussion………………………………………….………….……………………115 

3.4.1  Linking N-cycle genes, forage grass and N fertilization………………………...115 

3.4.2 N-cycle gene abundance and soil chemical characteristics……………………118 

3.4.3 Relationships of N2O emissions with N-cycle genes, N fertilization and soil 

properties………….……………………..………………………………………...119 

3.5….Conclusions…….....…………………………………………..…………………...120 

References…………..……………..….……………….………………………....122 



 
 

CHAPTER 4: BROMATOLOGICAL QUALITY AND ESTIMATED MEAT 

PRODUCTION IN MAIZE INTERCROPPING WITH TROPICAL FORAGE 

GRASSES WITH NITROGEN FERTILIZATION……………………………….131 

4.1      Introduction…………………………………………….…………………………..132 

4.2      Materials and methods……………………………….…………………………...133 

4.2.1   Study site…………………………………………………….……………………..133 

4.2.2   Study design………………………………………….……………………………134 

4.2.3   Crop management…………………………………………..…………………… 134 

4.2.4   Forage dry matter and maize grain yield……………………………………….135 

4.2.5   Bromatological analysis of forage grass……………………………….……….135 

4.2.6   Light interception, SPAD index and height of grasses………………………..136 

4.2.7   Estimated meat production………………………………………….…………...136 

4.2.8   Statistical analysis……………………………..………………………….………137 

4.3      Results………...……………………………………...…………………….……...137 

4.3.1 Forage dry matter and bromatological quality, and grain yield at maize 

harvest…..………………………………………………………………………….137 

4.3.2   Forage light interception and SPAD index in grass cut…………………………140 

4.3.3   Forage dry matter yield and bromatological quality at the final………………..140 

4.3.4   Estimated meat production…………………………………………...……..…...143 

4.4      Discussion…………………………………………………….……………………143 

4.4.1   Forage yield and bromatological quality yield at maize harvest………………143 

4.4.2   Light interception, height and SPAD index after maize-harvest……..………..145 

4.4.3   Dry matter, bromatological quality and estimated meat production………….146 

4.5   Conclusions.......................................................................................................147 



 
 

        References………..………………………...…………………………….……...….148 

        FINAL CONSIDERATIONS……………………………………………...………...155 

        REFERENCES……………………………..………………...……………….…….157 

  



 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



25 
 

GENERAL INTRODUCTION 

 Maize (Zea Mays L.) is among the most important food sources in the world, 

playing a key role (Ileri et al., 2018) in ensuring food security. It has been used for 

animal feed, human nutrition and, more recently, for bioethanol production (Ranum et 

al., 2014). As a C4 plant, maize is capable of achieving high dry matter yields 

accumulating large amounts of nutrients (Uzun et al., 2020). Usually, nitrogen (N) is 

the nutrient most required by maize (Teixeira et al., 2014; Wang et al., 2017). Nitrogen 

defficiency can limit crop yields, since this element is a constituent of important 

molecules such as amino acids, proteins, nucleic acids, nitrogenous bases, and 

chlorophyll (Moreira and Siqueira, 2002). One of the most studied topics regarding N 

in agricultural systems refers to strategies to improve N use efficiency (NUE) by crops, 

which can be achieved through conservation practices, like systems with different 

species intercropped (Adewopo et al., 2014; Rosolem et al., 2017). 

 The adoption of integrated systems, such as the intercropping of maize with 

forage grasses has been of great relevance for tropical agriculture, allowing the use of 

land throughout the year (Crusciol et al., 2009; Kichel et al., 2009). However, 

increasing the NUE through conservation systems is paramount, as it is necessary to 

ensure adequate soil N availability for plants and to reduce losses in the agricultural 

systems (Rosolem et al., 2017). The volatilization of NH3 from fertilizer sources is an 

important issue when urea is applied on the soil surface in no-till systems (Mariano et 

al., 2012), due to the higher activity of the urease enzyme when compared to 

conventional tillage (Silva et al., 2017). However, in Brazil,  with the N rates currently 

used and split applications, the risk of NO3
- reaching the groundwater is relatively low 

(Villalba et al., 2014). In addition, since N fertilizer is critical to sustain or increase the 

crop yield, the application of high N rates can lead to subsequent high N2O emissions 

in N-fertilized soils compared with those unfertilized (McSwiney and Robertson, 2005; 

Martins et al., 2015). No-till systems also largely affect the organic matter in the soil 

(Sá et al., 2015., Souza et al., 2016), and during the anaerobic decomposition of 

organic matter, CH4 production can occur (Dutaur and Verchot, 2007). Therefore, crop 

management strategies to decrease leaching losses and N2O emissions, as well as to 

increase NUE are fundamental for achieving adequate sustainability levels in 

agricultural systems (Rosolem, et al., 2017). 



26 
 

 Despite the several benefits from the no-till systems in relation to conventional 

cropping systems, such as improvements in the chemical, physical and biological 

properties of the soil, and reduction of CO2 emissions (Lal et al., 2007), maize-grass 

intercropped systems can result in competition between species for N, which can 

compromise crop yields (Borghi et al. 2014). The rotation and intercropping of maize 

with tropical grasses of the genus Urochloa (syn. Brachiaria) and Megathyrsus (syn. 

Panicum) is very common for integrated crop-livestock systems in tropical Brazil 

(Salton et al., 2014). In addition, it has been suggested that forage species of the genus 

Urochloa and Megathyrsus can affect microbiological processes within the N cycle, as 

well as N availability and losses (Subbarao et al., 2012). Subbarao et al. (2012) 

reported that biological nitrification inhibition (BNI) capacity was much higher in U. 

humidicola than in U. decumbens, M. maximus, Lolium perenne, U. brizantha, cereal 

and vegetable crops studied in a sand-vermiculite culture for 60 days. Thus, using 15N 

to study fertilizer N recovery by cash crops intercropped with forage species is 

fundamental (Rocha et al., 2019) to assess whether the fate of fertilizer N in the soil-

plant system is affected by BNI, when this process is active. 

 The N transformations in the soil by microorganisms can occur in several ways 

(Zhang et al., 2006), and the understanding of these processes is essential in the 

search for efficient and sustainable agricultural systems. In the atmosphere, N is found 

as a diatomic molecule (N2), which can be fixed by a specialized microbiota, by a 

process known as biological N fixation (BNF; Cardoso, 1992). In the soil, this element 

can be found in organic and mineral forms (Cantarella, 2007). The soil N dynamics is 

complex, mostly driven by soil microorganisms  as follows: i) BNF: the enzyme 

nitrogenase, which is encoded by the nifH gene, breaks the N2 triple bond to reduce 

N2 to ammonia (NH3; Zhang et al., 2006); ii) nitrification: NH4
+ is converted to NO3

- via 

the action of ammonia monooxygenase (amoA). This process comprises two phases: 

oxidation of NH4
+ to nitrite (NO2

-) and oxidation of NO2
- to NO3

-; iii) denitrification: 

copper nitrite reductase (nirK), iron nitrite reductase (nirS) and nitric oxide reductase 

(norB; Levy-Booth et al., 2014) oxidize NO3
- successively to NO2

-, NO and finally N2O; 

iv) the only route for converting N2O to N2 (Sun et al., 2019) is nitrous oxide reductase 

(nosZ; Henry et al., 2006). 

 Considering all these aspects, the following hypotheses can be formulated: 



27 
 

1. Forage species with high BNI capacity would increase maize grain yield and 15N 

recovery in the soil-plant system by suppressing soil nitrification and therefore 

decreasing fertilizer N losses; 

2. Tropical forage grasses would affect N cycle-associated genes and mitigate N2O 

emissions in N-fertilized maize; 

3. Nitrogen fertilization would increase maize yield, as well as dry matter yield and 

bromatological quality of forage grasses. 

Based on these hypotheses, the objectives of this study were: i) to estimate 

N2O, CH4, and NH3 emissions in maize-based rotation systems as affected by tropical 

forage grasses; ii) assess whether rotation with tropical forage grasses influences 

maize dry matter yield, N accumulation, 15N recovery, and the fate of the N fertilizer in 

the plant-litter-soil system over two growing seasons; iii) to characterize changes in 

total bacterial and archaeal abundances and in microbial populations involved in N-

fixation (nifH), ammonia oxidation (AOA and AOB), and denitrification (nirS and nosZ) 

and to measure N2O emissions in the maize intercropped with forage grasses; and iv) 

to assess maize grain yield and forage production, bromatological quality and 

estimated meat production in maize-forage grass intercropped systems. 

The first chapter of this thesis, entitled: “Fate of 15N fertilizer applied to maize in 

rotation with tropical forage grasses” was published in Field Crops Research. Chapter 

2, entitled: “Effect of tropical grass and nitrogen fertilization on nitrous oxide, methane, 

and ammonia emissions of maize-based rotation systems” was published in 

Atmospheric Environment; and the third chapter, entitled: “Functional N-cycle genes in 

soil and N2O emissions in a maize/tropical forage grasses intercropping system” was 

recently submitted to Science of the Total Environment. Lastly, chapter 4, entitled 

“Bromatological quality and estimated meat production in maize intercropping with 

tropical forage grasses with N fertilization” will be submitted in due course to Grass 

and Forage Science. 

 

 

 

 

 



155 
 

FINAL CONSIDERATIONS 

This study brings the importance of understanding some mechanisms that 

govern the integrated production systems such as intercropping and crop rotation in 

conservationist systems, through the choice of N fertilizer rates and forage grass 

species. These systems prove to be sustainable, since they allow the use of land 

throughout the year in addition to the recovery of degraded pastures. In addition, the 

discussions of the 21st century that address climate change across the planet (Oertel 

et al., 2016) show us the essentiality of studying systems that can result in more 

sustainable environments, aiming the reducing hunger in the world, by increasing 

production without increasing the area cultivated. In this way, the NUE, as well as the 

reduction of losses of this nutrient in the soil and in the atmosphere, become 

fundamental in the current agricultural systems, since N is one of the most limiting 

nutrients for plants (Teixeira et al., 2014; Wang et al., 2017). According to the results 

obtained, it was possible to understand the interactions between agricultural 

management and plant-soil-microorganism relationships can affect the N cycle 

(Bowles et al., 2013) and plant yield. Despite reports suggest that BNI by Urochloa 

spp. and Megathyrsus spp. decrease N loss in the system, it was not evident in N-rich 

environments. Regarding to maize rotation with forage grasses, there was no 

difference among the forage grass species in the distribution and fate of 15N in the 

plant–litter–soil system and, consequently, in unrecovered–N (i.e., potential losses). 

The amount of residual labeled N taken up by maize in the second growing season 

was very low. Guinea grass, palisade grass and ruzigrass did not affect N2O and NH3 

emission due to their apparent inability to suppress soil nitrification. However, N 

fertilization slightly increased cumulative N2O emission in the second maize season 

and decreased soil CH4 uptake in the fertilized palisade grass and ruzigrass relative to 

unfertilized palisade grass in the second forage season. 

Regarding the results of maize intercropped with forage grasses, cumulative 

N2O emissions were higher in the N-fertilized treatments than those without N addition. 

Moreover, a close relationship was observed among soil characteristics (NH4
+-N, NO3

-

-N, and WFPS), gene abundance (AOB) and N2O emissions. No relationship was 

found between denitrifying genes and N2O emissions. This suggests that although N 

modifies the soil microbiome, in addition to the abundance of AOB being highly linked 

to the increase in N2O emissions in the intercropped system, the increase in N-N2O 



156 
 

emissions to the system is small in relation to productivity gains of the system, 

suggesting a decreasing the specific emission, that is, the emission per unit of product.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



157 
 

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