JOSIANE APARECIDA VIVEIROS DE OLIVEIRA RECOVERY OF PHYTOTOXICITY INDUCED BY CARFENTRAZONE IN CORN AND COTTON CROPS BY FOLIAR SUPPLEMENTATION WITH MONOAMMONIUM PHOSPHATE Botucatu 2023 JOSIANE APARECIDA VIVEIROS DE OLIVEIRA RECOVERY OF PHYTOTOXICITY INDUCED BY CARFENTRAZONE IN CORN AND COTTON CROPS BY FOLIAR SUPPLEMENTATION WITH MONOAMMONIUM PHOSPHATE Tese apresentada à Faculdade de Ciências Agronômicas da Unesp Câmpus de Botucatu, para obtenção do título de Doutora em Energia na Agricultura. Orientador: Carlos Alexandre Costa Crusciol Coorientador: Caio Antonio Carbonari Botucatu 2023 A toda minha família Que são grandes incentivadores dos meus estudos, Dedico. AGRADECIMENTOS Começo agradecendo por sempre ter muita sorte, pois em todos os lugares que passei e me trouxeram até aqui, eu nunca estive sozinha, então a cima de tudo, agradeço a Deus, que me permiti caminhar com saúde, estudar, trabalhar, sempre aprendendo coisas novas e em todos os momentos rodeada de muitos amigos. Para falar das pessoas boas que aparecem no meu caminho, não poderia começar por outra, a não ser meu orientador e amigo Carlos Alexandre Crusciol. Professor, obrigada por confiar em mim desde o primeiro momento e por me permitir fazer parte do seu time. Eu sou imensamente grata por ter a oportunidade de trabalhar com uma pessoa de tão bom coração, além de toda inteligência e profissionalismo que nos serve de exemplo todos os dias. Quero agradecer também a toda a minha família, principalmente aos meu pais, Vera e Anísio, por serem minha base, por todo amor que tem por mim e nunca pouparem esforços para me verem crescer. Além da família de sangue, me considero privilegiada por ter amigos que se tornam família na minha vida. Muitos não estão presentes geograficamente, porém vocês todos estão sempre no meu coração e nas minhas orações. Começando lá atrás, obrigada a todo o meu time de basquete de Mirassol e Mari (Ralé), que foi quando aprendi o que era amizade de verdade. A todos os amigos que fiz durante minha faculdade: República Ibiza (em especial, Mene, Tuf, Milena e Corega); nossas lindas agregadas Manu, Roia e Fartura; às amizades que o esporte me trouxe mais uma vez: Amandinha, Aline e Mari (VL). Republica casa das Prima: Leitera, Annas e Tati e a toda República Litraço. Aos grandes amigos que fiz em Chapadão do Sul: Zefa, Explosão, Silvia Laguna, Bile (acelera) e Ana (Donana); E a todos os amigos que de alguma forma fizeram parte dessa etapa da minha vida: André, Tortuga, Por bosta, Marrone, Tirin, Arisca, Desmaiada, Pata, Palestrinha, My tropa (Boiada), Fuga, Flores, as irmãs Larissa e Carol, Purê, Rola, Biu, Litoral, Osama, Disk, Pula, Duelo, Meq, Gabrielona, Calmaria, Xuxu, Destronca, Julia, Mari (Ceri), Lais, Fafá e Caio. A toda república Renegadas, Saideira e Jaú Serve. É imensa a gratidão que sinto por ter vocês como parte da minha vida, espero que cada um de vocês saiba disso. Obrigada por tanto carinho, apoio, torcida e por fazerem meus dias melhores. Agradeço também a toda equipe de trabalho, principalmente Marcela e Luiz (Berim). Obrigada por toda parceria, comprometimento e paciência. A todos os amigos que trabalham no departamento e fazem com que trabalhar se torne mais fácil e agradável. A banca avaliadora pela disposição e tempo dedicado a esse trabalho. A todas as empresas que forneceram área e/ou produtos para realização deste projeto. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. Enfim, OBRIGADA! ABSTRACT Oxidative stress, whether biotic or abiotic, can cause severe damage to agricultural crops, including corn. The use of herbicides to control weeds, although efficient, can lead the plant to chemical stress, due to phytotoxicity to crops, significantly increasing the production of reactive oxygen species in plants (ROS) and directly and significantly affecting significant to the final productivity of the crop. In the case of the carfentrazone-ethyl herbicide, there is an accumulation of reactive oxygen species in the cytoplasm, which results in rupture of the membranes and the appearance of chlorotic spots on the leaves. Among the management practices studied to mitigate such effects, the use of nutrients via the leaves has gained space in this sector. This practice aims at specific nutrition for the plant, not in order to replace conventional fertilization, but rather as a supplement at a given moment in the development of the crop. Nitrogen and phosphorus perform important functions for plants, such as participating in photosynthesis and ATP formation, transport of assimilates, cell division, activation of the antioxidant system, directly affecting the final productivity of the crop, and when applied via the leaves, they enter directly in the metabolic processes of the plant, providing faster responses. Thus, in this study, the herbicide was applied to the corn crop, inducing stress on the plants, for subsequent application of MAP via the leaves in order to evaluate the effects of these nutrients on the recovery of the phytotoxicity of the crop. Plant nutritional status, gas exchange, pigments, photosynthetic enzymes, antioxidant metabolism, oxidative stress, amino acids, metabolites and biometric analysis were evaluated. Keywords: foliar fertilization; carfentrazone; elimination of ROS; productivity; antioxidant system. RESUMO O estresse oxidativo, seja ele biótico ou abiótico, pode causar danos severos nas culturas agrícolas, dentre elas o milho. O uso de herbicidas para controle de plantas daninhas, apesar de eficiente, pode levar a planta ao estresse químico, devido a fitotoxicidade às culturas, elevando de forma significativa a produção de espécies reativas de oxigênio nas plantas (EROs) e afetando diretamente e de forma significativa a produtividade final da cultura. No caso do herbicida carfentrazone-ethyl, ocorre o acúmulo de espécies reativas de oxigênio no citoplasma, que resulta em rompimento das membranas e o aparecimento de manchas cloróticas nas folhas. Entre os manejos estudados para mitigar tais efeitos, o uso de nutrientes via foliar, tem ganhado espaço neste setor. Essa prática visa uma nutrição específica para a planta, não a fim de substituir a adubação convencional, mas sim como uma suplementação em um momento determinado do desenvolvimento da cultura. O nitrogênio e o fósforo exercem funções importantes para as plantas, tais como participar da fotossíntese e da formação de ATP, transporte de assimilados, divisão celular, ativação do sistema antioxidante, afetando diretamente na produtividade final da cultura, e quando aplicados via foliar, entram diretamente nos processos metabólicos da planta, proporcionando respostas mais rápidas. Dessa forma, nesse estudo foi realizado aplicação do herbicida na cultura do milho, induzindo estresse sobre as plantas, para posterior aplicação de MAP via foliar a fim de avaliar os efeitos desses nutrientes na recuperação da fitotoxicidade da cultura. Foram avaliados estado nutricional das plantas, trocas gasosas, pigmentos, enzimas fotossintéticas, metabolismo antioxidante, estresse oxidativo, aminoácidos, metabólitos e análises biométricas. Palavras-chave: aubação foliar; carfentrazone; eliminação de EROs; sistema antioxidante; produtividade. FIGURES LIST CHAPTER 1 - INFLUENCE OF MAP FOLIAR APPLICATION ON RECOVERY FROM CHEMICAL STRESS IN CORN CROP Figure 1 - Precipitation and temperature history in Santa Cruz do Rio Pardo- SP, Brazil, during the two corn crop cycles, from January to June 2021e janeiro a junho de 2022.………………………………………………………………………..28 Figure 2 - Applications carried out during the conduction of experiments with the corn crop: Induction of phytotoxicity and foliar supplementation with P in V4, V6, V8 and R1. ..................................... …………………………………………………..30 Figure 3 - Nutritional status of maize plants, as indicated by N and P concentrations, as a function of MAP foliar fertilization. Different letters indicate significant differences between treatments by Fisher's protected Least Significant Difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). ....................................................................... ………………………...34 Figure 4 - Net photosynthetic rate (A, B), substomatal CO2 concentration (C, D), stomatal conductance (E, F). Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). .......................... …………………………………………………………..36 Figure 5 - Evapotranspiration (A, B), water use and efficiency (C, D), carboxylation efficiency (E, F). Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). .......................... …………………………………………………………..38 Figure 6 - Chlorophyll a (A, B) and chlorophyll b (C, D) rates as a function of foliar application of MAP to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). ................................................................. ………………………..39 Figure 7 - Rates of total chlorophylls (A and B), carotenoids (C and D) and rubisco activity (E and F), as a function of foliar application of MAP to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). .......................... …………………………………………………………..40 Figure 8 - Superoxide dismutase (A, B), ascorbate peroxidase (C, D) and catalase (E, F) as a function of foliar application of MAP to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4)…………………………….………………………………………………42 Figure 9 - Hydrogen peroxide (A, B), malondialdehyde (C, D) and proline (E, F) as a function of P foliar application to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4)…………………………………….…………..4444 Figure 10 - Sucrose (A and B) and starch (C and D) contents as a function of MAP foliar application to mitigate phytotoxicity effects. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). ....................................................................... ………………………...45 Figure 11 - Plant population (A, B), plant height (C, D) and number of branches (E, F) as a function of foliar application of MAP to mitigate phytotoxicity effects. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). .... …………………………………………………………………………..46 Figure 12 - Number of grains (A, B), mass of 100 grains (C, D) and grain yield (E, F) as a function of foliar application of MAP to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). …………………………….………………………………………………...48 CHAPTER 2 - FOLIAR APPLICATION OF MAP AS A WAY TO MITIGATE THE EFFECTS OF OXIDATIVE STRESS IN COTTON CROPS. Figure 1 - Average rainfall and temperature in Riolândia-SP, Brazil, during the two cotton crop cycles, 2021 and 2022. ……………………………………………………………………………….69 Figure 2 - Applications carried out during the experiments with the cotton crop: Induction of phytotoxicity in V4 and foliar supplementation with P in B1, F1, C1 and C4. ..................... ……………………………………………………………...71 Figure 3 - Nutritional status of cotton plants, as indicated by P concentrations (A, B), as a function of MAP foliar fertilization. Different letters indicate significant differences between treatments by Fisher's protected Least Significant Difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). ................................................... ……………………………….……….75 Figure 4 - Hydrogen peroxide (A, B), malondialdehyde (C, D) and proline (E, F) as a function of foliar application of MAP to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). …………………………………………………………….………………..76 Figure 5 - Superoxide dismutase (A, B), ascorbate peroxidase (C, D) and catalase (E, F) as a function of foliar application of MAP to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4)..…………………………………………………………………………….77 Figure 6 - Chlorophyll a (A, B), b (C, D) and total chlorophyll (E, F) rates as a function of MAP foliar application to mitigate phytotoxicity effects. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4)………………………………………………………...……………………79 Figure 7 - Carotenoids (A, B) and Rubisco activity (C, D) as a function of foliar application of MAP to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). ........................................................................ ………………………….80 Figure 8 - Sucrose (A, B) and starch (C, D) contents as a function of MAP foliar application to mitigate phytotoxicity effects. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4)……………………………………..……………………………………….81 Figure 9 - Plant population (A, B), plant height (C, D) and number of branches (E, F) as a function of foliar application of MAP to mitigate phytotoxicity effects. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). ……………………………………………………………………………….82 Figure 10 - Number of bolls (A, B), boll weight (C, D) and cotton yield (E, F) as a function of MAP foliar application to mitigate phytotoxicity effects. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). ......................................................................... ……………………84 Figure 11 - Strength (A, B) and fiber length (C, D), as a function of foliar application of MAP to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4)…………………………………………………..………………………….85 TABLES LIST CHAPTER 1 - INFLUENCE OF MAP FOLIAR APPLICATION ON RECOVERY FROM CHEMICAL STRESS IN CORN CROP Table 1 - Soil characterization in Santa Cruz do Rio Pardo, at a depth of 0-20 cm, in the 2021 harvest…….…………………………………………..……..……………..29 Table S1 - Nutritional status of corn plants indicated by concentrations of N, K, Ca, Mg, S, Fe, Cu, Zn, Mn and B as a function of foliar fertilization with MAP after stress induction. Different uppercase and lowercase letters indicate significant differences between treatments by Fisher's protected Least Significant Difference (LSD) test at p ≤ 0.10………………...…………………………..……………………………65 CHAPTER 2 - FOLIAR APPLICATION OF MAP AS A WAY TO MITIGATE THE EFFECTS OF OXIDATIVE STRESS IN COTTON CROPS Table 1 - Soil characterization in Riolândia, at a depth of 0-20 cm, in the 2021 harvest…………………………………….……………………………….70 Table S1 - Nutritional status of coton plants as indicated by N, K, Ca, Mg, S, Fe, Cu, Zn, Mn and B concentrations as a function of foliar fertilization with P after stress induction. Different lower, case letters indicate significant differences among treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.10..………………………………………………...…………………..…..94 SUMMARY GERAL INTRODUCTION .................................................................................. 21 CHAPTER 1 - INFLUENCE OF MAP FOLIAR APPLICATION ON RECOVERY FROM CHEMICAL STRESS IN CORN CROP............................ 24 1.1 Introduction .......................................................................................................24 1.2 Material and methods ........................................................................................27 1.2.1 Development of the experiment and description of the site ................................27 1.2.2 Experimental area and treatments .....................................................................29 1.2.3 Applications .......................................................................................................29 1.2.4 Management practices ......................................................................................30 1.2.5 Analyzes ............................................................................................................31 1.2.5.1 Nutritional analysis ............................................................................................31 1.2.5.2 Gas exchange and pigments .............................................................................31 1.2.5.3 Enzymatic activity ..............................................................................................32 1.2.5.4 Oxidative stress .................................................................................................32 1.2.5.5 Amino acids .......................................................................................................33 1.2.5.6 Metabolites ........................................................................................................33 1.2.5.7 Biometric Assessments .....................................................................................33 1.2.5.8 Statistical analysis .............................................................................................33 1.3 Results ..............................................................................................................34 1.4 Discussion .........................................................................................................49 1.5 Conclusion ........................................................................................................53 References ........................................................................................................54 CHAPTER 2 - FOLIAR APPLICATION OF MAP AS A WAY TO MITIGATE THE EFFECTS OF OXIDATIVE STRESS IN COTTON CROPS ......... 66 2.1 Introduction.......................................................................................................66 2.2 Materials and methods .....................................................................................68 2.2.1 Development of the experiment ........................................................................68 2.2.2 Description .......................................................................................................69 2.2.3 Experimental design and treatments ................................................................70 2.2.4 Application of treatments ..................................................................................70 2.2.5 Crop management ..........................................................................................71 2.2.6 Sampling, measurements and data analysis……………………………………….71 2.2.7 Nutritional analysis ............................................................................................72 2.2.8 Antioxidant metabolism and oxidative stress .....................................................72 2.2.9 Photosynthetic pigments and gas exchange ......................................................73 2.2.10 Photosynthetic enzyme and metabolites……………………………………………..73 2.2.11 Biometric Assessments………………………………………………………………...74 2.3 Results……………………………...……………………………………………………74 2.4 Discussion……………………………...………………………………………………..85 2.5 Conclusion………………..……………………………………………………………..89 References .. …………………………………………………………………………….91 FINAL CONSIDERATIONS.................................................................................95 REFERENCES…………………………………………………………………………97 21 GERAL INTRODUCTION In Brazil, the production of large crops is of paramount importance for the national scenario, and the estimated area increase for the 2022/2023 harvest is 3.3%, totaling 77 million hectares, which corresponds to an increase of 13. 8% or 37.5 million tons compared to the previous harvest. For corn and cotton crops, an increase of about 2.1 and 4.0% is estimated, respectively (CONAB, 2023). Although the estimates are positive in relation to the increase in grain production, several factors influence the development of crops and prevent them from reaching their maximum productive potential. These factors can be stresses of a biotic nature, such as microorganisms, insects and invasive plants or abiotic, which occur as a result of physical and chemical factors. (SHARMA et al., 2012;KAUR et al., 2019; SACHDEV et al., 2021). Plants have their own stress response mechanism and these systems are activated as they are exposed to certain conditions, activating this defense system which is divided into signal perception, responses at the molecular level and morphophysiological responses. (JOSEMARA; QUEIROZ, 2010). The use of high doses of herbicides or the derivation of these products in plants that are sensitive, can result in phytotoxicity in the crop, that is, it can lead the plant to a situation of abiotic stress, considerably increasing the formation of reactive oxygen species (ROs): superoxide (O2 -), singlet oxygen (1O2), hydrogen peroxide (H2O2) and the hydroxyl radical (OH-), which in excess result in lipid peroxidation and degradation of membranes (FARMER; MUELLER, 2013). O2, an essential element for plant metabolism, necessary for metabolic processes such as aerobic respiration, photosynthesis and photorespiration (BARBOSA et al., 2014), under stress conditions leads to the formation of reactive oxygen species, which occur mainly in mitochondria, chloroplasts and peroxisomes, and these processes result in harmful changes to plant growth and the consequent drop in productivity (BARBOSA et al., 2014). O2- is the first reactive species of O2 formed in the process of oxidative stress, it is considered an element of slow diffusion and little reactivity when compared to other forms of ROS. What makes the formation of O2- worrying is that if the process is not reversed in this phase, new species of oxygen, much more 22 reactive and harmful, are formed from this element, such as OH- and 1O2. The dismutation can occur spontaneously or through the antioxidant enzyme, SOD, which generates molecules as a final product. H2O2 and H2O. The OH- molecule can also be generated, because O2- can interact with Fe+ forming the hydroxyl radical and this reaction is called the Haber-Weiss reaction. Another reaction that can occur is Fenton's, the last step of the process being the oxidation of H2O2, in the presence of iron or copper, generating OH-1 (BARBOSA et al., 2014; BHATTACHARJEE, 2010; GILL; TUTEJA, 2010). The H2O2 it is the least reactive molecule among ROS and also the smallest in size, which enables its diffusion through membranes and this translocation allows hydrogen peroxide to play the role of messenger of biotic and abiotic stresses. This molecule, despite being little reactive, in high quantities can deactivate some enzymatic groups by oxidation and participate in reactions that result in the formation of OH-(KARUPPANAPANDIAN et al., 2011). The OH- is the last molecule formed by the Haber-Weiss reaction, that is, it occurs in the presence of Fe+ or Cu+ (BHATTACHARJEE, 2010; GADJEV; STONE; GECHEV, 2008; KARUPPANAPANDIAN et al., 2011) and cannot be controlled by any antioxidant system, as it is the most reactive molecule of the family, that is, the only way to control this species of oxygen is through the control of molecules that participate in reactions that have the hydroxyl radical as a product Final. This molecule is capable of inactivating or causing mutations in nitrogenous bases, it can denature proteins, damage carbohydrate molecules and give rise to lipid peroxidation leading to cell death. (BARREIROS; DAVID; DAVID, 2006a; BLOKHINA; VIROLAINEN; FAGERSTEDT, 2003). The 1O2 it is less reactive than the hydroxyl radical and its damage affects photosystems I and II. The little entry of CO2 in the system favors its formation, that is, the formation of singlet oxygen is favored by stomatal closure. Like the hydroxyl radical, this molecule cannot be removed by means of the enzymatic system, and to be eliminated it is necessary that there is a transfer of excitation energy from the 1O2 to other molecules, or being oxidized by organic molecules such as proteins, nucleic acids, lipids and carbohydrates (RONSEIN et al., 2006; HAVAUX, 2014). The production of ROS occurs naturally and is part of cellular aging, however under normal conditions, there is a balance between the production of 23 ROS and the antioxidant system, in this way the enzymes antioxidants can reduce the negative effects caused by these substances (TEIXEIRA et al., 2017). The production of ROS resulting from the contact between the carfentrazone-ethyl herbicide and the plants occurs as follows: In the chloroplast, the PROTOX-inhibiting herbicides prevent the transformation of protoporphyrinogen IX into protoporphyrin IX, with this an accumulation of protoporphyrinogen IX occurs in the chloroplast causing allowing it to diffuse into the cytoplasm. In the cytoplasm, protoporphyrinogen IX undergoes non-enzymatic oxidation, becoming protoporphyrin IX, which, in the presence of light, forms singlet oxygen, initiating the process of lipid peroxidation. That is, oxidation of proteins and lipids, which results in loss of chlorophyll, loss of carotenoids and membrane rupture, causing chlorotic spots, wilting and necrosis of affected plants within 2 days (DUKE et al., 1991; BERTUCCI; FOGLEMAN; NORSWORTHY, 2019a). Nutrition plays a key role in the defense system of plants, increasing their resistance to stress. (MARSCHNER, 1995) and favoring crop productivity. Among the essential nutrients, nitrogen (N) and phosphorus (P) stand out, macronutrients that limit crop growth (PAIVA et al., 2012). P can be considered an energy element for plants because it composes the ATP (adenosine triphosphate) molecule, therefore, responsible for plant growth. In this way, P contributes to the maintenance of membrane structures, synthesis of biomolecules, formation of high-energy molecules, cell division, enzyme activation/inactivation and carbohydrate metabolism. (MALHOTRA et al., 2018a). On the other hand, N is used in the synthesis of proteins, chlorophyll, nucleic acids, phytohormones and metabolites, being fundamental in plant metabolism and photosynthesis. (BANG et al., 2020) Knowing this, the two elements are of great importance in stress situations, as they enable new metabolic strategies that avoid overloading photosynthetic processes, control the overproduction of ROS, preventing damage to the plasmatic membrane (MALHOTRA et al., 2018a). Thus, this research tested the hypothesis that foliar supplementation with N and P in corn and cotton crops can mitigate the effects of oxidative stress in crops after contact with carfentrazone-ethyl herbicide. Thus, the objective of this work is to evaluate how the plant responds to the foliar application of MAP and 24 what is the best phase of the crop to apply it, in order to attenuate the symptoms of phytotoxicity. CHAPTER 1 INFLUENCE OF MAP FOLIAR APPLICATION ON RECOVERY FROM CHEMICAL STRESS IN CORN CROP Abstract The combination of the application of Nitrogen (N) and Phosphorus (P) through the leaves, with the time of application, can mitigate the effects of chemical stress. The objective of this work was to evaluate the impact of foliar application of N and P source (MAP) on the development and productivity of maize plants, under conditions of chemical stress. The herbicide used for stress induction was carfentrazone-ethyl, inhibitor of the enzyme protoporphyrinogen oxidase (PROTOX) and stress induction was performed when the plants were in the phenological stage V3. The experiments were conducted in Santa Cruz do Rio Pardo for 2 agricultural years and the design used was randomized blocks with seven treatments and four replications. Treatments consisted of: T1 - absolute control; T2 - control with stress induction; T3 – stress induction + P in V4; T4 – stress induction + P in V6; T5 – stress induction + P in V8; T6 – stress induction + P in R1; T7 – stress induction + P in V4+V6+V8+R1. In addition to the nutritional status of the plants, gas exchange, pigments, photosynthetic enzymes, antioxidant metabolism, oxidative stress, amino acids, metabolites and biometric analysis were evaluated. The results showed that foliar supplementation with P was efficient in mitigating the effects of oxidative stress caused by the herbicide, as it provided 100% recovery in crop development and productivity when spraying was performed at V4, V6, V8 and R1 (T7). Keywords: leaf supplementation; carfentrazone; elimination of ROS; Zea mays; productivity. 1.1 Introduction Brazil is the fourth largest grain producer in the world, with an estimated productivity of 312.5 million tons for the 22/2023 harvest in an estimated area of 77 million hectares, an increase of 3.3% compared to the previous harvest. The numbers point to an increase of 40.1 million tons compared to the 2021/22 harvest. 25 Due to the great economic importance of agricultural crops, and the need to produce more and more in the same area, there is a growing demand for alternatives that make this process possible. (CONAB, 2023). Weed control is fundamental for the good development of crops, as the presence of weeds in areas of agronomic interest can harm plants in nutritional and phytotechnical aspects. The control of these plants must be carried out aiming at efficiency and lower environmental impact, an objective that can be achieved through integrated weed management, which includes the combination of cultural, mechanical and chemical control methods. (MARTINELLI et al., 2017). Chemical control is carried out through the use of herbicides that can be applied before or after the emergence of the crop, and in general it must be done according to the level of interference they exert on the crop. To facilitate the understanding of the control periods Pitelli (1985) established the periods of coexistence between weeds and crops: Pre-Interference Period (PIP), which is the period between sowing and the beginning of weed interference, that is, when weeds do not present a risk of loss to the crop. culture; total interference prevention period (TIPP), the ideal period in which weeds must be controlled so that the crop can manifest its productive potential without interference; and critical period of interference (CPI) (PITELLI; DURIGAN, 1984; ANDREANIJUNIOR, 2016). The determination of these periods varies according to the crop in question, planting time, environmental conditions, weed community and production system employed. (BLEASDALE, 1960). Thus, the number of days within each period may vary: PIP: 15-20 (V2-V4), TIPP: 40-45 (V6-V8) and CPI: 15-45 (V2-V7) (PITELLI, 1985; (KOZLOWSKI, 2002; BALBINOT et al., 2016; SALGADO et al., 2002). Despite the advantages of this control method, the wrong use of these products can lead to problems, such as the risk of environmental contamination, selection of resistant weeds, intoxication of people and even plants, which, even though they are selective to certain herbicides due to their ability to to metabolize the products, they may still show symptoms of phytotoxicity (LANGARO et al., 2017). In situations where phytotoxicity occurs, there may be high production of reactive oxygen species (ROS). These oxygen species occur in plants that have suffered some type of stress, which can be biotic or abiotic, and can appear as superoxide anion (O2), hydrogen peroxide (H2O2), hydroxyl radical (OH-), singlet 26 oxygen (1O2), methyl radical (CH3-), and lipid peroxidation free radicals (LOO, ROO), which under normal conditions are related to plant development, but the excess produced under stress conditions impairs the redox balance, which regulates the plant's defense system and reactive oxygen species, causing damage to different parts of the plant, called stress oxidative (HAYAT; AHMAD, 2007; KIM; KWAK, 2010). Herbicides act on plants by altering or inhibiting functions that differ according to their mechanism and mode of action, which is nothing more than the primary site where the product acts and the sequence of events that occurs after the contact of the herbicide with the plant, respectively. The herbicide carfentrazone, belongs to the group of inhibitors of the protoporphyrinogen oxidase enzyme (PROTOX), which interrupts the formation of chlorophyll precursors and is focused on the control of broadleaf plants (DUKE et al., 1991; BERTUCCI; FOGLEMAN; NORSWORTHY, 2019b). In the chloroplast, PROTOX-inhibiting herbicides prevent the transformation of protoporphyrinogen IX into protoporphyrinogen IX. In the cytoplasm, protoporphyrinogen IX undergoes non-enzymatic oxidation, becoming protoporphyrin IX, which, in the presence of light, forms singlet oxygen, initiating the process of lipid peroxidation. That is, oxidation of proteins and lipids, which results in loss of chlorophyll, loss of carotenoids and membrane rupture, causing chlorotic spots, wilting and necrosis of affected plants within 2 days (DUKE et al., 1991; BERTUCCI; FOGLEMAN; NORSWORTHY, 2019b). In stressful situations, plants rely on their defense system to maintain cellular redox balance, which may be non-enzymatic, through ascorbic acid, vitamin E, flavonoids, proline, glutathione and other molecules. (DAVAR; DARVISHZADEH; MAJD, 2013), or of an enzymatic nature, such as the enzymes Superoxide Dismutase (SOD), Catalase (CAT) and Ascorbate Peroxidase (APX). However, this defense process is possible due to the presence of some nutrients that directly participate in the metabolic actions of plants. (MALAVOLTA; VITTI; OLIVEIRA, 1997). Thus, it is important to highlight that a well-nourished plant tends to show greater tolerance to stress (MARSCHNER, 1995) and, as the degradation of the membrane caused by PROTOX inhibitors occurs in plants affected by the herbicide, some nutrients have functions directly linked to the structure and integrity of membranes (TAKANO et al., 2019). 27 In addition to conventional fertilization, nutrients can be introduced into crops through foliar fertilization in order to provide plants with better resources in situations that are unfavorable to their development, where there is a greater nutritional demand. (FERNÁNDEZ; BROWN, 2013). It is already known that foliar- applied nutrients directly enter the metabolic processes, which provides faster responses when compared to the use of these nutrients in the soil, but the absorption and consequent efficiency of this application varies according to the nutrient, the characteristic of each plant. , environment and product used (FERNÁNDEZ; BROWN, 2013). Among the essential nutrients that benefit the recovery of plants in stress situations, nitrogen and phosphorus play a role from the activation of the antioxidant system, composing amino acids, chlorophylls, nucleic acids and ATP (MALHOTRA et al., 2018ª; Bang et al., 2020). Among the sources of N and P, MAP is used as a source of phosphorus and nitrogen and comes from the treatment of ammonia with phosphoric acid (REBELATO; PASSINATO; CAMPOS, 2018). Studies indicate that in direct contact with the leaf, the elements N and P can reduce symptoms of abiotic stress in corn plants when supplied in the vegetative stage, because, in addition to rapid absorption, they promote protection of the photosynthetic system and activate the defense system plant antioxidant (TAIZ; ZEIGER, 2017; REID, 1997). In this sense, this research will test the hypothesis that, if there is no self- recovery of the plants after phytotoxicity, the foliar application of MAP will provide positive responses to the plants, favoring their development and directly reflecting on productivity. Thus, the objective of this work is to evaluate how the plant responds to the foliar application of MAP and what is the best phase of the crop to apply it, in order to attenuate the symptoms of phytotoxicity. 1.2 Material and methods 1.2.1 Development of the experiment and description of the site The study was carried out in 2 harvests (2021 and 2022) and conducted in the region of Santa Cruz do Rio Pardo - SP. 28 The coordinates of the location are (22° 50′ 7″ S, 49° 31′ 09.4″ W, altitude 467 m above sea level). The climate in the region is long, hot and muggy summer, dry, short winter and the temperature is rarely below 11°C. On average, the annual temperature is around 15 to 31°C and precipitation is approximately 1236.5 mm during the year. The average precipitation and temperature at the site, during the two years of the experiment, can be seen in Figure 1. Figure 1 - Precipitation and temperature history in Santa Cruz do Rio Pardo- SP, Brazil, during the two corn crop cycles, from January to June 2021e janeiro a junho de 2022. Source: own authorship Table 1 shows the values obtained in the soil analysis carried out at the site. 29 Table 1 - Soil characterization in Santa Cruz do Rio Pardo, at a depth of 0-20 cm, in the 2021 harvest. Soil classification Riolândia (LVdf)a Climate (koppen-Geiger)b Cwa pH (CaCl2) 5.5 MO (g dm-3) 21 P (mg dm-3) 28 S (mg dm-3) 2 Al+3 (mmol dm-3) 0 H+Al+3 (mmol dm-3) 25 K (mmol dm-3) 3.3 Ca (mmol dm-3) 55.2 Mg (mmol dm-3) 11.9 SB (mmol dm-3) 60 CTC (mmol dm-3) 85 V (%) 70 m (%) 0 Fe (mg dm-3) 19 Cu (mg dm-3) 10.4 Mn (mg dm-3) 12.0 Zn (mg dm-3) 2.5 B (mg dm-3) 0.42 1.2.2 Experimental area and treatments The experiments were conducted in randomized blocks, with 4 replications and 7 treatments: 1 - absolute control; 2 - induction of phytotoxicity; 3 - induction of phytotoxicity + application of phosphorus in V4; 4 - induction of phytotoxicity + application of P in V6; 5 - induction of phytotoxicity + application of P in V8; 6 - induction of phytotoxicity + application of P in P in R1 and 7 - induction of phytotoxicity + application of P in V4, V6, V8 and R1. Plots consisted of 11 m x 7 rows with 0.45 m spacing between rows. 1.2.3 Applications Phytotoxicity induction was induced using carfentrazone-ethyl herbicide, which belongs to the group of PROTOX inhibitors. The dose for inducing phytotoxicity was 50 ml i.a.. ha-¹ + 0.5% of mineral oil, and the MAP dose used for foliar application was 5 kg ha-¹, which corresponds to 3.1 kg of P2O5 ha-1 and 0.55 30 kg ha-1 in NH4 + The dose used to induce phytotoxicity was established after tests with different doses of the product in the culture, while the MAP dose was determined based on research carried out in the literature. All sprays (Figure 2), both herbicides and MAP, were carried out with the aid of a knapsack sprayer with constant pressure (CO2), equipped with a 3 m bar with 6 fan-type tips (AXI 11002) spaced at 0.50 m. The volume of syrup used was 150 L ha-1, pressure of 1.80 bar. Figure 2 - Applications carried out during the conduction of experiments with the corn crop: Induction of phytotoxicity and foliar supplementation with P in V4, V6, V8 and R1. Source: own authorship 1.2.4 Management practices The corn hybrid used was P3707VYH DuPont Pioneer, with a population of 3 m-¹ seeds. Seeds were treated with carboxin + tyrant fungicides (100 g + 100 g a.i. 100 kg-¹). Corn plants were fertilized with 280 kg ha-¹ of 28-08-16 in the sowing furrow. Subsequently, 172 kg ha-¹ of urea and 25 kg ha-¹ of potassium were manually applied at the V4 stage of the crop. 31 1.2.5 Analyzes For purposes of nutritional analysis, gas exchange, oxidative stress, antioxidant metabolism, amino acids, pigments and photosynthetic enzymes and metabolites, leaf samples were collected when the plants were in the R2 phenological stage (white grains with the appearance of a water bubble ) (FEHR et al., 1971; HANWAY, 1963). 1.2.5.1 Nutritional analysis For nutritional sampling of macro and micronutrients nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca) and sulfur (S) and boron (B), iron (Fe), zinc (Zn), manganese (Mn) and copper (Cu), respectively, the first leaf below the first ear was collected, totaling 10 leaves per plot and using the middle third of each leaf. After collection, the leaves were dried and placed in an oven with forced air circulation at 65°C for 72 hours. The material was then ground in a Willey mill in a sieve with a mesh of 1 mm in diameter to evaluate the nutritional contents of macro and micronutrients, according to the methodology described by Malavolta, Vitti and Oliveira (1997). 1.2.5.2 Gas exchange and pigments For the determination of net photosynthesis (A) (µmol. m-² s-¹), stomatal conductance (gs) (mol m-² s-¹), carbon concentration in the substomatal chamber (Ci) (µmol mol-¹), transpiration (E) (mmol m-² s-¹), carboxylation efficiency (A/Ci) and water use efficiency (A/E). Data were collected using the IRGA (Infra Red Gas Analyzer), model CIRAS-3 by PP Systems, which performs all the measurements mentioned above. For the three harvests, readings were taken on 5 plants per plot in the morning, between 9:00 am and 11:00 am, with a constant ambient CO2 of 390 µmol mol-¹. The photosynthetic pigments chlorophyll a, chlorophyll b, total carotenoids and total chlorophylls were determined by cutting five discs of 0.5 cm in diameter, between the edge and midrib, of the last fully expanded leaf. The samples were stored for 24 hours in glass vials containing 2 mL of N, N-dimethylformamide (DMF) and wrapped in aluminum foil according to the methodology proposed by 32 Lichtenthaler (1987). Pigment contents were quantified using the spectrophotometric method at wavelengths of 664, 647 and 480 nm for chlorophyll a, b and carotenoids, respectively. Pigment concentrations were determined as proposed by Wellburn (1994). 1.2.5.3 Enzymatic activity The total Rubisco activity was measured according to the method described by Reid et al. (1997). Frozen plant material (0.3 g) was ground with a mortar and pestle under liquid nitrogen and suspended in extraction buffer containing 1.5 mL of 58 mM potassium phosphate and 1 mM ethylenediaminetetraacetic acid (EDTA). The homogenized material was centrifuged at 14,000 rpm for 25 min at 4°C and the supernatant was stored at 4°C (adapted from Sage et al., 1988; Reid et al., 1997). Rubisco incubation buffer contained 100 mM bicine-NaOH pH 8.0, 25 mM potassium bicarbonate (KHCO3), 20 mM of magnesium chloride (MgCl2), 3.5 mM ATP, 5 mM phosphocreatine, 0.25 mM NADH, 80 nkat glyceraldehyde-3- phosphate dehydrogenase, 80 nkat 3-phosphoglycerin phosphokinase and 80 nkat creatine phosphokinase. A 70 μL aliquot of the supernatant was incubated with 900 μL of the incubation buffer at 30°C for 5 min in the absence of ribulose-1,5-bisphosphate (RuBP) to allow for Rubisco carbamylation. NADP oxidation was initiated by adding 30 µL of 16.66 mM RuBP directly to the cuvette. Readings were obtained in a spectrophotometer at a wavelength of 340 nm. Rubisco activity was calculated from the difference in absorbance readings at 0 and 1 min (without removing the cuvette from the spectrophotometer) and expressed in μmol min–1 mg protein–1. 1.2.5.4 Oxidative stress Collections took place in the morning, between 9:00 am and 11:00 am and were quickly stored in falcon tubes, placed in liquid N and stored at -80°C. The contents of hydrogen peroxide (H2O2), malondialdehyde (MDA), as well as superoxide dismutase (SOD; EC:1.15.1.1), catalase (CAT; EC:1.11.1.6), ascorbate peroxidase APX (EC:1.11.1.11) were evaluated for the three cultures. Lipid peroxidation was evaluated according to the method of Heath e Packer, 1968. To calculate the MDA content, a molar extraction coefficient of 155 mM 1 cm 1 was 33 used and the results were expressed in nanomoles of MDA per gram of fresh weight (FW). The H2O2 content was determined according to the method of Alexieva et al., 2001 and the content was calculated based on a calibration curve and expressed in µmol g−1 FW. SOD activity was evaluated as described by Giannopolitis and Ries, 1977, and the results were expressed in units (U) of SOD per milligram of protein. CAT activity was evaluated as described by Azevedo et al., 1998 and results were expressed in micromoles per minute per milligram of protein. APX activity was evaluated according to Gratã et al., 2008, and the results were expressed in nanomoles per minute per milligrams of proteine 1.2.5.5 Amino acids The proline content for the three crops, was determined according to Torello e Rice, 1986. The absorbance at wavelengths of 647 and 664 nm was determined using a spectrophotometer, and the results were expressed per gram of FW, as described by Mauad et al., 2016. 1.2.5.6 Metabolites The same leaves collected to assess the nutritional status were used to analyze the reducing sugar, total sugar, starch and sucrose content. (NELSON, 1944; SOMOGYI, 1945). 1.2.5.7 Biometric Assessments Upon reaching physiological maturity, the height of 10 plants per plot were measured, from the apex to the base, and 10 ears per plot were collected to determine the number of rows per spike, number of grains per row and weight of 100 grains. In addition, the number of plants per hectare was stipulated, and for productivity, the useful area harvested was 3 m from the two central lines of each plot. Moisture was corrected to 13% and the weight was later converted into bags per hectare. 1.2.5.8 Statistical analysis 34 Data were submitted to analysis of variance (ANOVA) and, when significant, the means were compared by the LSD test, at a 10% probability level, using the statistical program Sisvar®. 1.3 Results There was no significant difference between treatments for leaf N and P content, which can be seen in Figure 3. Figure 3 - Nutritional status of maize plants, as indicated by N and P concentrations, as a function of MAP foliar fertilization. Different letters indicate significant differences between treatments by Fisher's protected Least Significant Difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). Source: own authorship MAP applications at different phenological stages influenced gas exchange parameters in maize (Figures 4 and 5). In the first and second harvests, the application of MAP provided recovery in the photosynthetic rate of the plants with 11.2 and 26.1%, respectively, in relation to the control (22.8 µmol CO2 m−2 s−1) and there was no significant difference between the treatments that received MAP, regardless of the time of application (Figure 4A and 4B). In the second year, there was a significant difference between treatments for Ci (internal carbon concentration), where treatments that received MAP application had a lower 35 concentration of Ci, with a difference of up to 33.9% in relation to C-Phyto (162.5 μmol mol-1) (Figure 4D). The stomatal conductance increased with the application of P in corn, with a difference of 14.1% in the first year and 16.0% in the second year of the treatments that received P in relation to C-Phyto, where averages of 208 and 130.8 mol were observed. H2O m-2 s-1 in the first and second year (Figure 4E and F). 36 Figure 4 - Net photosynthetic rate (A, B), substomatal CO2 concentration (C, D), stomatal conductance (E, F). Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). Source: own authorship Leaf transpiration was higher in the treatment with carfentrazone application and without supplementation with MAP (C-Phyto), with 2.6 and 5.7 mmol H2O m-2 37 s-1, differing significantly from the treatments, which showed lower values of up to 13.0% in the first year and 26.6% in the second (Figure 5A and B). Water use efficiency (WUE) and carboxylation efficiency (Ce) were significantly affected by P foliar applications, with values higher than C-Phyto in the first and second harvests for both variables (Figure 5C, D, E and F). For WUE there was an increase of 27.4 and 63.8% in relation to C-Phyto (8.7 and 4.94 µmol CO2 (mmol H2O)-1, while for Ce, in the second year, there was an increase of 100% of P-All.Appl when compared to C-Phyto (Figure 5F). 38 Figure 5 - Evapotranspiration (A, B), water use and efficiency (C, D), carboxylation efficiency (E, F). Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). Source: own authorship Photosynthetic pigments and Rubisco were influenced by MAP foliar applications, regardless of maize phenological stage (p<0.05) (Figure 6 and 7). 39 Overall, the lowest concentrations of photosynthetic pigments were observed in C- Phyto. Figure 6 - Chlorophyll a (A, B) and chlorophyll b (C, D) rates as a function of foliar application of MAP to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). Source: own authorship The same trends were observed for the total content of chlorophylls and carotenoids, since regardless of the time of application, treatments with P supply differed significantly from C-Phyto, with an average of 20.5% for total chlorophyll and 37.3% for carotenoids (Figure 7). Treatments that received foliar P after stress induction with carfentrazone differed significantly from C-Phyto (33.5 and 61.3 mmol H2O2 min-1 g FW-1), with increased rubisco activity by up to 32.8 and 16.3% in the first and second year, respectively (Figure 7E and F). 40 Figure 7 - Rates of total chlorophylls (A and B), carotenoids (C and D) and rubisco activity (E and F), as a function of foliar application of MAP to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). Source: own authorship All parameters related to oxidative stress and antioxidant metabolism (H2O2, MDA, SOD, CAT, APX and Proline) were significantly influenced (p<0.1) 41 by the foliar application of MAP in the two corn crops (Figure 8 and 9). Overall, among the treatments that received P application, P-All.Appl. had less accumulation of SOD, APX and CAT in both seasons, reducing on average 20.2 and 17.3% for SOD, 38.9 and 21.4% for APX and approximately 74 and 25% for CAT in relation to C-Phyto, which presented, respectively, during the two years, 153 and 212.5 U SOD g FW-1min-1, APX 29 and 59.5 mmol H2O2 min-1 g FW-1 and CAT 1.05 and 1.68 mmol H2O2 min-1 g FW-1 (Figure 8). 42 Figure 8 - Superoxide dismutase (A, B), ascorbate peroxidase (C, D) and catalase (E, F) as a function of foliar application of MAP to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). Source: own authorship Treatment 2 (C-Phyto) also differed significantly from the other treatments, with the exception of proline in the second year (Figure 9F), where there was no significant difference between treatments. The treatments that received foliar supplementation with MAP decreased the levels of H2O2 significantly, in the first 43 and second year (Figure 9A and B), when compared to C- Phyto (4.24 and 6.7 nmol H2O2 g FW-1) with P-All.App there was a reduction of 18.4 and 33.6%. The observed valuesfor MDA, 10.4 4 12.5 nmol MDA g FW-1 were reduced by up to 29.7% in P-R1 and 24% in P-All.App, in the first and second year, however, overall, there was no difference between treatments that received MAP, regardless of the time of application. In the first year the proline contents in P-All.App. significantly reduced compared to C-Phyto (0.86 umol g-1 FW), by 30.2%. 44 Figure 9 - Hydrogen peroxide (A, B), malondialdehyde (C, D) and proline (E, F) as a function of P foliar application to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). Source: own authorship There was a significant difference (P<0.1) in the first year for sucrose between treatments that received P when compared to C-Phyto (1.35%). 45 Treatments demonstrated, on average, a 68% increase in sucrose content after MAP application (Figure 10A). The concentration of starch in C-Phyto (2.03 and 3.18%) differed significantly from the other treatments in the two years, being on average 28.4% and 19.5% higher in relation to the treatments that received MAP application (Figure 10 C and D). Figure 10 - Sucrose (A and B) and starch (C and D) contents as a function of MAP foliar application to mitigate phytotoxicity effects. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). Source: own authorship There was no significant difference (P<0.1) between treatments for population, height and number of branches in the second year (Figure 11). In the 46 first year, only for plant height, there was a difference between treatments, on average, the All treatment. differed significantly from the others with an increase of up to 7.6% in relation to the smallest treatment (154.3 cm). Figure 11 - Plant population (A, B), plant height (C, D) and number of branches (E, F) as a function of foliar application of MAP to mitigate phytotoxicity effects. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). Source: own authorship 47 P-All.Appl. demonstrated greater recovery of the culture with values close to C.A. and differed significantly from the other treatments in number of grains per row and weight of 100 grains in the first year and grain yield in both harvests (Figure 12). For the number of grains in the first year, there was an increase of 5.13% when compared to C-Phyto (25.5 grains) (Figure 12A), while in the second, all treatments that received MAP differed from C-Phyto (27.3 grains) with increment of, on average, 3.07% (Figure 12B). With the same trend, for a weight of 100 grains in the first year, the treatments that received P showed an average increase of 8.36% in relation to C-Phyto (19.0 g) (Figure 12 C). In the second year P-All.Appl differed significantly from the other treatments with an increase of 11.2% in relation to C- Phyto (24.0 g) (Figure 12 D). Supplementation with MAP proved to be efficient for the final productivity of the crop. P-All.Appl provided a gain of 13.5% in the first year and 11.04% in the second, in relation to the C-Phyto treatment, where productivity of 4,135.75 kg ha- 1 in the first year and 6,403.50 kg ha-1 in the second agricultural year (Figure 12E and F). 48 Figure 12 - Number of grains (A, B), mass of 100 grains (C, D) and grain yield (E, F) as a function of foliar application of MAP to mitigate effects of phytotoxicity. Different letters indicate significant differences between treatments by Fisher's protected least significant difference (LSD) test at p ≤ 0.1. Error bars express the standard error of the mean (n = 4). Source: own authorship 49 1.4 Discussion Leaf supplementation did not result in a significant difference between treatments for N and P content. This may occur due to the small concentration of nutrients provided, linked to good soil fertility during the conduct of the experiment, in this way the nutrients supplied enter into a dilution effect, due to the high mobility when in direct contact with the leaves. This effect is linked to an external factor that promotes a high dry matter index. (MARSCHNER,1995). The term stress can be defined as adverse conditions unfavorable to the development of plants and are divided into two large groups: biotic stress, related to pests, diseases and invasive plants, and abiotic stress, caused by non-living factors, such as water, thermal or even chemical (BRIDGEMOHAN; MOHAMMED, 2019). In response to these effects, plants have developed a multitude of forms of defense against one or more types of stress that can affect them throughout their cycle, which may occur at the molecular and cellular level. (NEJAT; MANTRI, 2017). Plant responses to abiotic stresses are shown mainly in changes in chlorophyll content, osmotic adjustment (NEJAT; MANTRI, 2017), early stomatal closure and activation of antioxidant enzymes (ALMESELMANI et al., 2006). Any of these processes, when altered, result in drops in productivity (DOS SANTOS et al., 2015) (BANERJEE; ROYCHOUDHURY, 2019). Knowing that carfentrazone- ethyl interrupts the synthesis of chlorophyll a, preventing the conversion of protoporphyrinogen IX into protoporphyrin IX, a significant drop in pigment contents (chlorophyll a, b and carotenoids) after stress induction was expected (HAVAUX, 2014). Supplementation with foliar N and P provided recovery of chlorophyll a, b and carotenoids in plants. This can be understood as these elements strengthen your defense system, fighting reactive O2 species produced by chemical stress. (BARREIROS; DAVID; DAVID, 2006b); AHN et al., 2005), reestablishing the metabolic processes, and consequently, the formation of pigments (LI et al., 2021). The stress induced by carfentrazone-ethyl significantly influenced physiological attributes, resulting in a decrease in A (net photosynthesis), gs (stomatal conductance), WUE (water use and efficiency) and A/Ci (carboxylation efficiency) and an increase in E (evapotranspiration) and Ci (internal carbon 50 concentration) due to the rapid stomatal closure that the plant presents in response to stress situations to avoid water loss (TAIZ; ZEIGER, 2017). Os resultados corroboram com (WICHERT; TALBERT, 1993) who observed a reduction in the same parameters due to the application of a PROTOX-inhibiting herbicide in soybean plants. Similar results were reported by Barbosa, 2022, where orange seedlings showed a decrease in gas exchange rates after induction of chemical stress. In other studies, Ahmad et al., (2021) analyzing corn plants and Pieters and El Souki (2005) evaluating rice plants, it was found that plants under water stress conditions showed the same trends in photosynthetic parameters.This is because after the application of a PROTOX-inhibiting herbicide, water deficiency may occur in the cells (MOSTOWSKA, 1998). This occurs in adjacent cells due to the rupture of the membrane of the cells affected by the herbicide, causing a decrease in the osmotic potential, which results, before the loss of chloroplast integrity and the appearance of necrosis, in rapid stomatal closure and a decrease in the photosynthetic rate. (CARRETERO, 2008) Leaf supplementation with MAP promoted recovery in photosynthetic parameters, which may be related to the large participation of N and P elements in the photosynthetic processes, since they are fundamental for the synthesis of ATP, NADP and NADPH, which will be used in the photosynthetic cycle (MIFLIN; LEA, 1976; HARPER, 1994), thus, according to Chapin et al., 1987 and Lambers, et al., 1989, the higher the N concentration in the leaf, the higher the plant's photosynthetic rate. Pi is also essential for the transport of trioses from the chloroplast to the cytosol, preventing the photosynthetic rate from being reduced due to the accumulation of carbohydrates in the chloroplast. (KUWAHARA; SOUZA, 2009), Rubisco activity in the second year did not show significant differences between treatments, while in the first year the induction of phytotoxicity significantly decreased the activity of the enzyme, a fact that may be related to the drought in the first year that subjected the crop to water stress, in addition to chemical stress. However, the application of MAP foliar increased the enzymatic activity, regardless of the time of application. This is because the increase in Pi availability resulting from the extra supply of phosphorus can increase the entry of Pi into the 51 chloroplast, which can induce an increase in the assimilation of CO2 (PARRY et al., 2002). Overall, antioxidant enzyme activity increased significantly after herbicide stress induction in the treatment that did not receive MAP supplementation. The results corroborate with Ahmad et al. (2021), who observed an increase in the concentrations of antioxidant enzymes in corn plants under conditions of saline stress. In general, the production of ROS is part of the natural process of plant growth, but a high production results in damage such as damage to membranes, lipids and proteins. (AHMAD et al., 2021). MDA is formed from lipid peroxidation, which makes it an indicator of oxidative stress and explains a higher concentration in plants after the induction of chemical stress. As well as the concentration of H2O2, a molecule formed from the dismutation of the superoxide anion (O2 -), which can occur naturally or through the enzyme superoxide dismutase, generating H2O2 and H2O (BARBOSA et al., 2014). Supplementation with MAP, regardless of the time of application, reduced the rate of enzymatic activity as the indicators of oxidative stress also decreased. Similar results were observed by Zhao et al. (2022), with supply of N and P in alfalfa plants under stress conditions by herbicides and Li et al., 2018, who verified an increase in the activity of antioxidant enzymes in melon plants under P deficiency and the increase in these enzymes proportional to the supply of the nutrient. Under stress conditions, there is an increase in antioxidant compounds, which interact with each other or individually to limit the generation of peroxidized by-products more effectively. (HERNÁNDEZ; MUNNÉ-BOSCH, 2015). Proline is an amino acid that also acts as an antioxidant removing reactive oxygen species, and its accumulation reflects the stress condition induced in maize plants. As with other stress indicators, proline concentration was higher in plants that received herbicide application and did not receive MAP supplementation. In studies, Hasan et al. (2022), also observed an increase in proline concentrations in weeds after herbicide application. Under stress conditions, proline can move from the cytoplasm to the chloroplast and detoxify the hydroxyl radical. (SIGNORELLI et al., 2014), therefore, as plants showed recovery from stress, there was a decrease in proline concentration. After the induction of phytotoxicity, there was a reduction in the % of sucrose and an increase in the % of starch, however, supplementation with MAP attenuated 52 the symptoms of intoxication, promoting a higher concentration of sucrose and a decrease in starch production. What determines the production of sucrose or starch is the availability of Pi, so that a high availability of Pi benefits the synthesis of sucrose, while the low availability of this element favors the storage of starch (reserve carbohydrate) (MALHOTRA et al., 2018b). In general, the number of plants in the second year was greater than in the first, which may be a consequence of the long dry period in 2021, a fact that may also explain the significant difference for the plant height variable, where only the treatment 7 was, on average, significantly higher than the others. The obtained results demonstrated that the stress caused by carfentrazone- ethyl herbicide in the corn crop significantly reduced the 100-grain mass and the final productivity of the crop. The results are linked to the fact that the development of corn plants was impaired due to the damage that phytotoxicity caused to the crop, that is, the accumulation of reactive oxygen species caused damage to the membranes, causing cell death and influencing the process of photosynthesis, which is directly linked to the reduction of chlorophylls, proteins and stomatal conductance (ARIF et al., 2020). The final production of a crop is dependent on its photosynthetic capacity, since grain filling depends on the accumulation of carbohydrates generated in photosynthesis. (WULLSCHLEGER; OOSTERHUIS, 1990), thus oxidative stress causes a reduction in final productivity (CRAFTS-BRANDNER; PONELEIT, 1992). Plant recovery was observed, in terms of productivity, after foliar supplementation with MAP, mainly in treatment 7. The greater availability of N and P favored the increase of photosynthesis and, consequently, greater grain productivity, in agreement with Hernández and Munné-Bosch (2015), Zhang et al. (2018) Dalirie, Sharifi and Farzaneh (2010), respectively. The great demand for N at the beginning of the development of the corn crop is one of the reasons that makes this element one of the greatest limiting factors in productivity. Thus, the N provided together with P, in the foliar application of MAP, may also have favored greater formation and grain filling, since the nutrient in direct contact with the leaf participates directly in the metabolic processes, resulting in greater productivity (HARPER, 1984; CARVALHO et al., 2001; LARA CABEZAS et al., 2004). 53 1.5 Conclusion Leaf supplementation with MAP attenuated the effects of chemical stress on corn plants and provided greater yield in crop productivity. The nutrients in the leaves potentiate the metabolic processes of plants and stimulate photosynthesis and carbohydrate production, acting positively on the antioxidant defense system of plants, providing greater protection to cells. In this way, the productivity parameters are also improved, presenting a higher grain yield. 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