Relationships of FY in oil palm with environmental variables were verified in this work. The incidence of FY increased over the years evaluated, and its progress was evident in all plots of the two farms, in different percentages depending on the plot. In addition, incidence class intervals above 10% increased in 2014 and 2015 in the analysis of new cases of the disease. This result is similar to those from studies on the progression of the incidence of FY found in the literature. Bergamin Filho et at. 12 analyzed cumulative FY incidence curves constructed with monthly and annual data, noting an increase in the incidences in the proportions of 0.40, 0.15 and 0.016 for plantations of 19, 14 and 9 years, respectively. As in the present study, there were plots with up to 50% accumulated incidence, and the economic viability of planting was impaired, so clarifying the cause of this disease is essential to establish management practices. Therefore, an analysis of environmental variables in Pará State, a region where the occurrence of this disease is common, and the plant's nutrition was performed to determine their correlation with FY.
The northern region of Brazil has the ideal climatic conditions for oil palm cultivation, as it requires some specific edaphoclimatic variables for satisfactory production. The ideal precipitation regime for this crop is an average annual rainfall of 1.800 mm. Another requirement is solar radiation. The necessary insolation to reach the productive potential of the oil palm is approximately 1.800/h/year, with a minimum of 5 h/day. In addition, greater productivity occurs in regions with small variations in temperature and annual averages between 25 and 27°C 40. However, these same factors promote several phytosanitary problems. According to the results obtained, higher precipitation intensity results in greater relative humidity of the air (83 to 89%), less evapotranspiration, and greater accumulation of water in the soil; these conditions and lower wind speeds provide a greater probability of FY occurrence. Thus, although oil palm requires 150 mm of precipitation per month, in this region, it can receive to 500 mm/month in the first half of the year. In months of the year with strong wind conditions (2 to 2.5 m/s), which is associated with high insolation values (220 to 250 h/month), i.e., reduced cloud cover and reduced rainfall and temperature (28°C), there was less progress of the disease. Consequently, with greater insolation and presence of winds, greater evaporation and reduction in the amount of water in the soil occurs. The importance of water balance has also been studied by Venturieri et al. 9, who showed that it has a negative correlation (p = 0.0002) with fatal yellow. In regions with mild water deficiency, no cases of this disease were observed.
In this work, the correlation of climatic variables with the incidence of FY was verified. There was a correlation between only actual evapotranspiration and wind speed in 2013 and 2014. However, when the data were lagged by thirty days immediately prior to the assessment of the disease, all climatic variables correlated with FY in 2014, indicating the importance of this approach. The lag in climatic data shows that a time interval is necessary for the plant to develop symptoms. Therefore, the manifestations of physiological imbalance in the field occurred due to intense rain and high relative air humidity thirty days immediately before the evaluations. To verify the morphological changes of water stress in the physiology of oil palm seedlings, Rivera-Mendes et al. 41 conducted experiments with four water conditions for 60 days: moderate deficit, field capacity, and partial and continuous flooding. Seedlings under permanent flooding had a 22% reduction in biomass when compared to that of the control, due to the higher rates of leaf respiration and limitations in the absorption and transport of macronutrients. Plants under partial waterlogging showed growth similar to that observed under optimal soil moisture conditions.
In addition to the relationship with climatic variables, the correlation of fatal yellow with soil fertility and plant nutrition was studied. The disease had a positive correlation with Fe, K and Mn found in the soil. Thus, there was an increased incidence in places with higher levels of these minerals in the soil. This positive relationship may have occurred due to deficiency in the absorption of the roots or the imbalance of these cations when compared to other cations in the soil. The nutrients P, Ca, Mg and B had a significant negative correlation, i.e., the disease decreased with increases in these elements in the soil. Seeking to understand the role of other nutrients in soil with FY (FY?), Silveira et al. 42 carried out an experiment with the omission of the macronutrients nitrogen, phosphorus, potassium, calcium, magnesium and sulfur and the micronutrients boron, iron, manganese, copper, zinc and molybdenum. These authors observed an increase in symptoms when there was omission of micronutrients other than zinc and less incidence in treatments with omission of macronutrients other than Ca and S. Several studies on nutrition and fertilization have been carried out to verify the relationship between mineral elements and FY. Research has not verified the simultaneous effects of soil class, soil fertility and plant nutrition. When fertilization was studied, foliar analysis of the nutrients was not performed. Therefore, it is not possible to determine if the nutrients were absorbed or if their lack was associated with the disease. In studies analyzing the mineral nutrients in the plant, soil analysis was not carried out to determine whether deficiencies in a certain nutrient in plants was caused by low concentrations in the soil or due to inadequate absorption, including that possibly due to excess water in the soil, which would cause anaerobic conditions for the roots. Therefore, studying plant nutrition and soil fertility simultaneously is essential to understand the plant's physiological processes.
As for nutrition, palm trees showed deficiency in the nutrients N, P, Ca, Mg, S, Zn and Cu and exhibited excesses of K and B. According to Guzmán 37, AUDPC had significant negative correlations with the nutrients N, Ca, Si, B and Fe and a positive correlation with K. The minerals P, Ca and Mg also had low levels in the soil, which is a possible explanation for their deficiency in plants. The N, S, Si, Fe, and Mn were present in adequate quantities in the soil. However, in the sampled plants, they were below the nutritional levels suitable for the crop. Among these elements, N and S are structural constituents of plant cell components, including chlorophyll, amino acids, nucleic acids, coenzyme A, S-adenosylmethionine, biotin, vitamin B1 and pantothenic acid. Therefore, their deficiencies inhibit plant growth, causing chlorosis and yellowing of the leaves 23. Iron deficiency is common when water saturation occurs in the soil because this causes anaerobiosis in the root system. Its characteristic symptoms are chlorosis between the ribs. The relationship between iron and FY was investigated by Viégas et al. 43 with four treatments of different doses of ferrous sulfate. Afterwards, the iron contents in the oil palm leaves were measured, they caused no reduction or increase in symptoms in the analyzed plants.
Silicon, on the other hand, is not an essential nutrient for many plant families. However, some species accumulate substantial amounts of this element in their tissues and exhibit enhanced growth, fertility and stress resistance when supplied with adequate amounts of this element. Thus, plants deficient in silicon are more susceptible to tipping and fungal infection. Freitas et al. 26 determined the role of silicon in reducing the severity of yellow Sigatoka (Mycosphaerella musicola) in banana trees grown in nutrient solution. The treatments had five concentrations of silicic acid (H4SiO4): 0. 0.5, 1.0, 1.8 and 3.6 mmol/L. The AUDPC for plants in the 3.05 mmol/L H4SiO4 treatment was 49.27% lower than that for plants without supplementary H4SiO4. In contrast, plants grown in a 3.6 mmol/L solution of H4SiO4 showed 23.53% more Si content in the leaves than plants grown without H4SiO4 supplementation. Thus, Si could be used in disease management.
Approximately 65% of oil palm trees had excess K and Mg deficiency. Potassium is the second most required nutrient in plants, and in the soil solution, it appears as the ion K+, which is absorbed in the roots of plants. In addition, K+ permeates plasma membranes, making it easily absorbed and transported in xylem and phloem. However, at high concentrations of this nutrient, the absorption of Ca2+ and Mg2+ is reduced by competitive inhibition 25. Mg deficiency induced by excess K in fertilization is common in crops such as banana and coffee, as they require large amounts of K 26. Mg is present in the chlorophyll molecule. In addition, it participates in enzymatic activation, acting as a cofactor for phosphorylative enzymes by forming a bridge between ATP or ADP pyrophosphate and the enzyme molecule. Therefore, deficiency in this element is characterized by yellowing of the older leaves 23.
In the soil classes PACal md/arg, PACd md/arg and PACal + PACd md/arg, with the highest amount of clay, there was a higher frequency of plots with FY incidence. The textural class is determined by the particle size distribution and affects other physical properties, such as drainage and water retention, aeration and soil consistency. The texture and structure of the soil define the surface area and porosity, which are the main factors associated with the storage and availability of water in the soil and the drainage of water from the surface to deep layers of the soil profile (Costa et al. 2014). Silveira et al. 42 analyzed the physical and chemical properties of 19 pedological profiles distributed in different plots of crop plantations. The authors classified the soil as a medium-textured yellow Latosol and showed the presence of compaction (1.365 kg/m³ to 1619 kg/m³) at depths of 30 and 60 cm. Thus, saturation of the soil in the superficial layer occurred during the period of greatest rainfall in the year, causing oxygen deficiency in the superficial layer of the soil. These conditions can be harmful to oil palm, causing root rot, due to its root system being dominantly distributed horizontally close to the soil surface. Regarding water in the soil and drainage, Bernardes 45 measured the water potential in the soil with a tensiometer with low soil aeration with values between 0 bar and 0.075 bar, while 0.075 bar and 0.30 bar are more suitable for the plant. Thus, areas at risk of flooding, which is related to the soil class, can cause anoxia of the oil palm's superficial roots, triggering nutritional deficiency and weakening the plant's resistance, making them more susceptible to infections by pathogens.
The proteomes showed proteins related to both biotic and abiotic stresses, especially in response to hypoxia, flooding and oxidative stress, upregulated in FY plants. Considering the studies and the lack of consensus regarding the origin of this disease, the identification and quantification, as well as understanding the implications of the differential abundances of these proteins in the metabolism of the plants affected by FY, becomes fundamental to suggest this origin.
Reactive oxygen species (ROS) are related to the regulation of signaling pathways and initial responses that occur to environmental stresses of biotic or abiotic origin 46, 47. Antioxidant system-related proteins like peroxidase 2 and 1-Cys peroxiredoxin (Fragment) were upregulated in plants with FY and Asy plants with little difference in their abundance. In this case, oxidative stress can be an indication of stress from soil flooding in the area of plant cultivation.
In the data, primary metabolism-related proteins involved in energy production were also identified as having little difference in abundance between conditions. Despite this, they were overall more accumulated in Asy plants.
Additionally, with regard to energy metabolism-related proteins, alcohol dehydrogenases were identified in the roots of FY and Asy plants. Alcohol dehydrogenases are involved in alcohol fermentation during hypoxia or anoxia. There is a decrease in energy production through oxidative phosphorylation. Fermentative metabolism promotes energy compensation through recycling of NAD + to the glycolytic pathway28. Increases in the glycolytic pathway and alcohol fermentation-related proteins has been identified as a key response of plants to flooded soils 48, 50. Adaptive mechanisms to handle oxygen deprivation include synthesis induction of a set of approximately 20 proteins known as anaerobic stress proteins (ANPs), which in addition to alcohol dehydrogenase, also includes carbohydrate metabolism-related proteins, such as those identified in this study 28. In this context, the results indicated that the analyzed plants were likely subject to hypoxic conditions, which were probably caused by soil flooding in the crop area before or during the sampling period of the roots analyzed in this study.
It is important to highlight the identification of flooding stress-related proteins, such as polygalacturonase 30, 51, that was identified with no significant differential abundance comparing the proteomes of plants with or without FY (Dataset Sx). However it was downregulated in plants with FY. This protein acts on the degradation of the cell wall, which leads to the formation of lysigenous aerenchyma49., Aerenchyma channels formation can also occur by ROS-induced programmed cell death50, which in the roots can provide flooding tolerance by improving oxygen and nutrient uptake under hypoxic conditions51. In maize submitted to flooding stress, the increase in polygalacturonase expression and formation of aerenchyma is related to an adaptive response and allows greater toleranceto flooding 51, 52. Therefore, the high levels of these proteins in plants with and without symptoms may be related to the occurrence of flooding in these environments. Although not significant, the higher levels of polygalacturonase in asymptomatic plants may be related to their possible tolerance to flooding.
Among the proteins upregulated in plants with FY are calcineurin B-like protein 10 (Q7FRS8) and steroid 5-alpha-reductase DET2 (Q2QDF6), which have been attributed to the response to starvation. Calcineurin B-like protein 10 acts as a Ca+ sensor in cell signaling in response to environmental stimuli such as salinity, osmotic stress and nutrient deprivation, among others, including response to pathogens52. This protein is involved in the activation of cellular responses from its binding and activation of serine/threonine-protein kinase52. Serine/threonine-protein kinase was also upregulated in plants with FY. Caltractin (P41210) has also been identified in plants with FY, being known as Ca-binding and signaling proteins in response to environmental stimuli53. Regarding steroid 5-alpha-reductase DET2, this protein acts in the biosynthesis of steroids like brassinosteroid, which is involved in the response to many environmental stresses54. However, the sequence identified in this dataset was assigned only to starvation. The higher levels of these proteins in plants with FY also suggests the weakness of these plants, including nutritional deficiency and infection by pathogens due to their likely susceptibility.
Under nutrient deficiency conditions, plants need to increase their efficiency in nutrient storage and transport. Although the nodulin-like protein is well known for its characterization in plant roots in symbiotic association with microorganisms, studies have highlighted the importance of this protein in the transport of nutrients and other molecules to favor the growth and development55 of even non-nodulating plants56. Considering the response to abiotic stimuli, the nodulin-related protein 1 (Q9ZQ80) identified in this dataset was attributed to the osmotic stress and response to cold and heat. However, the possibility that it is also contributing to the increase in nutrient transport efficiency in FY plants cannot be excluded.
The translationally-controlled tumor protein (TCTP, Q9ZSW9) was attributed only to the term water deprivation. On the other hand, in addition to being involved in water transport, studies have shown that the increase in levels of this protein is related to photosynthesis and fatty acid metabolism in response to environmental stresses57. TCTPs have also been considered important in the defense response to fungal infections in plants58. Therefore, the higher levels of these proteins in plants with FY may include responses to several environmental stresses, including the increase in infections by pathogens.
Defense response proteins were also identified in plants with and without FY symptoms. In symptomatic plants, five fungal response proteins were upregulated, while four were downregulated. Regarding proteins in response to bacteria, only two were upregulated in plants with FY, while three were downregulated. The little difference between the abundance of fungal or bacterial response proteins observed in the protein profiles of healthy and affected plants does not allow us to deduce that fungal or bacterial infection is the major factor by which plants develop FY symptoms. Furthermore, asymptomatic plants showed higher levels of proteins related to the response to nematodes and insects. In general, asymptomatic plants seem to respond better to different types of environmental stresses, which should be attributed to their healthy state or the difference in genotypes that should be studied in the future.
We cannot yet assure why some genotypes have not yet developed the symptoms of FY. On the other hand, considering the results obtained in the analysis of climate variables, soil classification and fertility, plant nutrition and the protein profiles of plants with FY symptoms, it is possible to suggest the flooding as one of the factors for the deficiency in the nutrients N, P, Ca, Mg, S, Zn and Cu in the observed plants, which may be associated with the greater susceptibility of these plants.