Application of exogenous compounds increased the tolerance to aluminum toxicity and improved fruit production of oil palm (Elaeis guineensis Jacq.) grown on Ultisols

The low productivity of oil palm grown on suboptimal land Ultisols with high Al saturation requires efforts to improve cultivation techniques in order to increase plant tolerance to Al stress and to improve fruit production. This study aimed to determine the effect of various exogenous compounds on physiological changes and fruit production of oil palm (Elaeis guineensis Jacq.) grown on Ultisols with high Al saturation. This study consisted of 2 treatments, namely: (1) a combination of various exogenous compounds, and (2) application methods. The experiment was arranged in RCBD split plots with 3 replications. Treatment of exogenous compounds was (1) control (untreated); (2) CaCl2 50 ppm; (3) CaCl2 50 ppm + Salicylic acid 50 ppm; (4) CaCl2 50 ppm + Salicylic acid 50 ppm + citric acid 50 ppm; (5) CaCl2 50 ppm + Salicylic acid 50 ppm + citric acid 50 ppm + boron 50 ppm; and (6) CaCl2 50 ppm + Salicylic acid 50 ppm + citric acid 50 ppm + boron 50 ppm + Cu 50 ppm. The application method used was (1) basal drench and (2) foliar spray. Application of exogenous compounds was carried out every 3 months. The results revealed that application of exogenous compounds has enabled plants to increase the tolerance to Al stress, as evidenced by an increase in the number of normal bunches and bunch weight, the increase of which was due to the decrease in the number of rejected bunches, especially in the CaCl2 + salicylic acid treatment, which was selected as the most effective treatment. Increased fruit production in this treatment was associated with improvements in physiological processes; decreased MDA and increased photosynthetic rate and activity of APX and CAT enzymes resulted in improvement fruit production. These positive changes in physiological processes after treatment suggested that the addition of those exogenous compounds has induced signalling processes involved in ROS scavenging, that promoted the tolerance to Al stress. Basal drench application method was more effective than foliar spray, this was related to the increase in soil pH and P available due to the application of exogenous compounds through basal drench method.


Introduction
Oil palm crops in Indonesia are predominantly cultivated on marginal lands so that their production is below their potential.Mineral acids soils (Ultisols) are one of the suboptimal soil types that are widespread in Indonesia.Land in Indonesia is dominated by acidic mineral soils reaching 74.6% [1].Low pH levels with high Al saturation followed by low nutrients are the main characteristics of this type of soil [2].The results of soil surveys in oil palm plantations, especially PT Waru Kaltim Plantations, East Kalimantan, showed that the dominant soil texture of sandy minerals with low pH and KTK and Al-dd 4.13 cmol(+)/Kg so that aluminum saturation is very high (>60%) [3].Although in general oil palm is relatively tolerant of Al depression as [4] stated that oil palm is able to accumulate Al in the roots up to 600 ppm.However, some studies state that Al toxicity will cause plant growth and development to be inhibited.Negative effect of Al toxicity on leaf area, plant height and total dry weight of seedlings [5].In 2020, the achievement of oil palm production in Jambi province, which is predominantly cultivated on Ultisols, was only 87% of the achievement of national production [6].Soil ameliorant applications are still a solution in the improvement of acidic lands such as the use of lime and dolomite [7] which require high inputs per area and costly.
Al-stress plants will carry out a tolerance mechanism naturally but slowly so there is a need for stimulants.The use of various exogenous compounds is able to induce plant tolerance to abiotic stress as a short-term problem solving [2].Various exogenous compounds have been shown to be able to induce transduction signals in plants that contribute to changes in gene expression in regulating the increased activation of some antioxidant enzymes such as CAT, APX and GPX.These enzymes are very important in protecting cells from oxidative damage by reactive oxygen species (ROS).Free Ca 2+ in the cytoplasm acts as a second messenger in the signal transduction process through binding by calmodulin for the defense of plant cells to Al stress [8].The involvement of calcium in increasing plant tolerance to the action of metals such as aluminum [9].In addition, Al stress can be reduced by the application of citric acid in cucumber plants [10], salicylic acid in wheat [11], soybeans [12].Generally, the application of exogenous compounds for Al stress is carried out on planting media, but some studies state that the application of exogenous compounds through leaves can increase plant tolerance to abiotic stress.Some of these exogenous compounds are organic acids in alfalfa [13], a mixture of semisynthetic chitosan derivatives [14] and calcium [15] in maize crops, in oil palm has not been found.This study aimed to determine the influence of various exogenous compounds on physiological changes and production of oil palm (Elaeis guineensis Jacq.) grown on Ultisols soils with high Al saturation.

Materials and Methods
The research was conducted at PT Waru Kaltim Plantation, East Kalimantan, Indonesia.The average temperature, annual rainfall and relative humidity are 25 • C, 1962 mm/year and about 75-85%, respectively.The plants used in this experiment were the 8-year-old Socfind variety, which was grown on Ultisols with a pH of 4.48, exchangeable Al 8.03 cmol(+)/kg and a saturation of Al 82.1%.The experiment used a RCBD of a two-factor split plot group.As the main plot was the treatment method of application, namely basal drench (BD) and foliar spray (FS); as a sub-plot was a combination of various exogenous compounds.The combination treatments of exogenous compounds include (1) control (untreated); (2) CaCl2 50 ppm; (3) CaCl2 50 ppm + Salicylic acid 50 ppm; (4) CaCl2 50 ppm + Salicylic acid 50 ppm + citric acid 50 ppm; (5) CaCl2 50 ppm + Salicylic acid 50 ppm + citric acid 50 ppm + B 50 ppm; and (6) CaCl2 50 ppm + Salicylic acid 50 ppm + citric acid 50 ppm + B 50 ppm + Cu 50 ppm.The application of exogenous compounds was carried out once every 3 months from January 2018 to October 2019.The basal drench (BD) method was performed by sprinkling exogenous compound around oil palm weeding circle 0.5 to 2 m away from the tree using a 10 L solution per experimental unit.The foliar spray (FS) method was conducted by spraying the oil palm leaves using a mist blower with a volume of 5 L of solution per experimental unit.Soil chemistry changes that observed were soil pH, P available, exchangeable Al and Al saturation.Physiological analysis was performed for MDA levels, activities of APX and CAT antioxidant enzyme at 3 DAT (days after treatments) and 7 DAT.Photosynthesis rate was measured from the 17 th leaf by CI-340 Handled Photosynthesis System at 3 DAT, 7 DAT and 45 DAT.

Soil Analysis
Soil samples were taken on the weeding circle of oil palm samples 1.5 m away from the palm trees with a depth of 0 to 30 cm using a mineral soil drill type belgi, then the samples were dredged and sent to the laboratory.Soil chemical variables observed were soil pH (potentiometric method), P (Bray-II method), Al (NHOAC, pH 7.0 method).

MDA levels, CAT and APX activities
The activity of MDA and CAT were determined by method developed by Chen and Zang [16].APX activity was determined by method developed by Nakano and Asada [17].Samples were taken from the 17 th leaf at 3 DAT and 7 DAT.

Productivity
This observation consists of the number of normal bunches, the number of rejected bunches and the average bunches weight were measured once a week.Rejected bunches are abnormal bunches that have a low percentage of fruit sets (< 25%) observed on each sample subject.Weighing of harvested fresh fruit bunches using digital scales to obtain data on the average bunches weight.

Data Analysis
Analysis of variance was carried out with the "F" test.Duncan's Multiple Range Test (DMRT) at 5% probability level was applied to estimate the difference in significance between treatments.Statistical program Genstat v19.1.0.21390 was used for data analysis while graphs were reproduced with Microsoft Excel 2010.

Soil pH, Exchangeable Al, Al Saturation and P Available
The results showed that the pH value of the Ultisols without treatment was 4,5 and exchangeable Al 6,6 cmol(+)/kg and Al saturation 77.3% (Table 1).Treatment of exogenous compounds with basal drench application increases soil pH even though it is relatively small.The soil pH value greatly affects the solubility of aluminum in the soil.The results of soil analysis revealed that exchangeable Al and Al saturation in treatments 2, 3, 4, 5 and 6 decreased compared to controls except foliar spray (FS) treatment (Table 1).The decrease in exchangeable Al in the soil was followed by an increase in P available to plants as seen in the treatment of exogenous compounds with basal drench application (Table 1).
The amount of P available in the soil was influenced by the exchangeable Al, because aluminum will bind P so that it is not available to plants.According to Chen and Liao [18] Al will bind P to form the Al-P complex so that it is not available to plants.The decrease in exchangeable Al and the increase in P available was predicted to be the influence of Al chelating by citric acid and salicylic acid as well as root exudate organic acids forming the Al-AO (organic acid) complex.Plant defense mechanisms to Al stress will secrete organic acids such as citric, oxalic and malic.The main organic acids secreted by the roots of Al-stressed plants are citric, oxalic and malic as Al-chelators [19].Organic acid secretion through the transduction signal pathway played by Ca 2+ in cytosols activates calmodulin which is related to the activation of protein kinase so as to regulate the expression of organic acid transporter genes.The complex process is very dependent on environmental conditions so exogenous organic acids are needed to stimulate the induction of plant tolerance.Plants are able to exclude Al from the tip of the root through the secretion of various organic acids where 60 -80% to exclude Al so as to avoid Al contamination [20][21].Apart from being an Al chelate, [12] pointed out that salicylic acid also plays a role in stimulating antioxidants so that lipid peroxidase is slowed down.

Photosynthesis Rate
The results showed that the treatment of exogenous compounds affects the rate of photosynthesis.In Al-stressed land, the photosynthesis rate of control plants at 3 DAT reached 8.1 μmol/m 2 /s.The treatment of exogenous compounds 2, 3, 4, 5 and 6 on a BD increases the rate of photosynthesis at 3 DAT, but application via FS has no effect.A similar pattern was seen in 7 and 45 DAT, treatments 3, 4, 5 and 6 by BD increased the rate of photosynthesis, but various treatments with the FS method had no effect (Table 2).Based on these results, it is clearly seen that salicylic acid and citric acid exert a significant positive influence on the increase in the rate of photosynthesis.According to [22][23][24] the application of citric acid was able to increase the rate of photosynthesis on Al-stressed plants.Salicylic acid plays an important role in maintaining the integrity and function of photosystems in photosynthesis, which promoted plant tolerance to Al stress [25].The numbers followed by the same letters in the same column show no significant difference based on DMRT at the level α = 5%; DAT: day after treatment;  1B) and APX (Figure 1C) activities at 3 DAT, the same pattern was also observed at 7 DAT (Figure 2A), which was followed by an increase in the activity of CAT (Figure 2B) and APX enzymes (Figure 2C).The foliar spray method of application was less effective than basal drench.There was a clear physiologically linkage, addition of exogenous compound reducing MDA content, and increasing the activity of APX and CAT enzymes, which promoted an increase in the rate of photosynthesis (Table 2).Plants exposed to oxidative stress will experience of lipid peroxidation resulting in accumulation of MDA.The presence of cell damage can be predicted by the amount of MDA accumulation in the tissue [26].However, plants have a complex mechanism of ROS scavenging in which several antioxidant enzymes play a role in neutralizing the effects of free radicals such as APX and CAT [27].Some literature revealed that various exogenous compounds have been shown to increase the activity of antioxidant enzymes, i.e. boron [28], salicylic acid [29], citric acid [22] [24] and calcium [30].[31] exhibited that exogenous citric acid is able to increase the activity of antioxidant enzymes and reduce MDA content in Al-stressed Pinus massoniana.The increased of POD and CAT activity after treated by exogenous citric acid, and concluded that citric acid has contributed to diminished the accumulation of MDA and H2O2 levels, leading to reduce a damage to cell membranes [22].Besides a citric acid, [12] mentioned that salicylic acid also plays a role in stimulating antioxidants so that lipid peroxidase was slowed down.This was in agreement with our work on oil palm seedlings exposed to Al stress under nutrient culture [24].  3 revealed a decrease in the number of rejected bunches due to the applications of exogenous compounds in all treatments compared to controls, the decrease of which was followed by an increase in the average bunches weight (Table 3).The basal drench method was more effective than foliar spray.The highest average bunches weight and productivity was obtained by treatment 3 with the BD method of 12 kg and 164.5 kg per palm per year, respectively.The increase in average bunches weight and decrease in the number of rejected bunches was related to the improvement in soil chemical properties such as increased pH, availability of P nutrients accompanied by a decrease in Al saturation, presumably addition of exogenous compound has involved tremendously in promoting fruit production especially under BD application.Moreover, the efforts to increase the rate of photosynthesis need to be carried out because it is closely related to the achievement of high productivity [32][33].A decrease in MDA levels accompanied by an increase in CAT and APX activities will increase the rate of photosynthesis which is thought to play a role in fruit formation so as to increase the number of normal bunches, average bunches weight (ABW) and plant productivity.The numbers followed by the same letters in the same column show no significant difference based on DMRT at the level α = 5%;

Conclusions
The presence of positive changes in plant physiological processes after treatments suggests that the addition of those exogenous compounds has induced signaling processes involved in ROS scavenging, which promoted the tolerance to Al stress and plant productivity.The basal drench application method is more effective than foliar spray.It is associated with an increase in soil pH and P available due to the application of exogenous compounds through the basal drench method.

Table 1 .
Effect of exogenous compounds on pH, exchangeable Al, Al saturation and P available

Table 2 .
Effect of exogenous compounds on photosynthesis rate

Table 3 .
Effect of exogenous compounds on number of rejected bunch, number of normal bunches, average bunch weight and productivity of oil palm aged 9 years