Effects of Two Chelating Agents on Availability of Calcium and Phosphorus in Black Soil of Vegetable Fields

Dai Jian-jun, Wang Xiao-chun, Fang Qiu-na, Liu Hong-fei, and Liu Li-zhi

College of Resources and Environmental Sciences, Northeast Agricultural University, Harbin 150030, China

Abstract: To explore the effects of the two chelating agents on the availability of calcium and phosphorus in black soil of vegetable fields and the growth of cabbage (Brassica pekinensis Rupr.) seedlings, humic acid (F) and ethylene diamine tetraacetic acid disodium salt (EDTA) were used as experiment materials, and two factors and three levels completely randomized experiment of the nine treatments was set. The experiments were carried out by soil incubation experiment and pot experiment to investigate the effects of the two chelating agents with different ratios and their interaction on soil and cabbage. The results showed that there were very significant differences among soil pH, soil available phosphorus content, cabbage biomass, calcium and phosphorus accumulation of cabbage under the interaction of the two chelating agents. It could improve pH of the soil and reduce the electric conductivity (EC) value of soil, when applied chelating agents of different proportions. The EC value of soil fluctuated during the incubation period. There were significant differences between pH and EC in the early incubation period, but there was no significant difference in the later stage. And there were significant differences between soil available calcium and available phosphorus content; there were significant differences between cabbage biomass and the accumulation of calcium and phosphorus in cabbage, and the data of F2E2 treatment were significantly higher than those of other treatments. In summary, under the conditions of the experiment, the available phosphorus content of black soil in vegetable fields, the biomass and accumulation of calcium and phosphorus in cabbage increased significantly in F2E2 treatment (appied 675 kg · hm-2 of lime, 300 kg · hm-2 of humic acid and 150 kg · hm-2 of EDTA).

Key words: humic acid, EDTA, soil calcium, soil phosphorus, cabbage seedling

Introduction

In northeast China, garlic (Allium sativum) is planted in spring and then cabbage (Brassica pekinensisRupr.)is replanted after garlic harvesting in late summer in the vegetable fields of black soil region, but cabbage root is often swollen in a large area. Chinese cabbage root disease is mainly caused by soil acidification and the decrease of soil calcium availability, which reduces the disease resistance of Chinese cabbage,therefore, plasmodiophoa brassicae woron infects the cabbage roots. However, the cabbage has being continuously planted for several decades, and the large demand of Ca2+in garlic and Chinese cabbage and excessive dose of nitrogen fertilizer applied by farmers result in soil acidification and excessive soil NH4+, inhibiting the absorption of Ca2+. Furthermore,excessive nitrogen fertilizer leads to excessive salt content in soil, which also inhibits plants absorption of nutrients, such as Ca2+. Calcium and phosphorus in vegetable fields of black soil area are often produced insoluble Ca-P, of which calcium and phosphorus availability both reduce simultaneously. Calcium deficiency in vegetables may often be accompanied by symptoms of phosphorus deficiency. For example,the phosphorus-deficient plant of Chinese cabbage is dwarfed, its growth rate is slow and the leaf color becomes darker, which seriously affects its quality and yield. Therefore, it is of great significance to study how to improve the availability of calcium and phosphorus in black soil of vegetable fields.

The application of calcium fertilizer in vegetable fields can improve vegetable quality and increase vegetable yield significantly (Jiaet al., 2015) and also promote root growth (Wanget al., 2012). The application of calcium fertilizer can increase the calcium content of Chinese cabbage, thus, effectively reducing the incidence of cabbage heartburn and root disease (Liet al., 2013). At the same time, studies have shown that calcium ions have a significant effect on regulating plant cells to salt damage (Ma and Zhao, 2006 and Debickaet al., 2016). The experiment applied an appropriate amount of lime to the soil to increase soil calcium content and also adjusted soil pH (the amount of lime was obtained by the amount of soil applied in the field). Phosphorus also plays an important role in vegetable growth (Kanget al.,2011). Studies have shown that phosphate fertilizer can promote the growth of plant roots and improve the utilization and transformation of nitrate nitrogen in plants (Ma, 2014). It is not uncommon to use chelating agents to improve the availability of soil calcium and phosphorus. Crops can absorb effective nutrients more effectively in chelated fertilizers by ion exchange. Humic acid is the main component of soil organic matter (Weiet al., 2007), with a variety of reactive functional groups (Lianget al., 2012; Wanget al., 2018). It is a ruthenium molecular organic acid composed of carbon, chlorine, oxygen, nitrogen,sulfur and other elements. Humic acid has various functional groups which can chelate with metal ions,such as iron, aluminum, calcium, magnesium, etc. in the soil. Humic acid easily coagulates with calcium ions in soil and then forms soil agglomerate structure through physiological action of plant roots, so that it can increase soil porosity and permeability. At the same time, humic acid can increase the content of organic matter, provide favorable conditions for the construction of micro-ecological environment, which is beneficial to the absorption of nutrients and normal growth of crops. Different components and ratios of chelating agents have significantly different roles and effects on the growth of crops (Liet al., 2015; Hanet al., 2009). Liu (2004) thought that humic acid could form a phosphohumic acid complex with insoluble phosphide to activate insoluble phosphate and further improve the utilization rate of phosphate fertilizer. Humic acid can also chelate with metal ions, compete with phosphate for soil adsorption sites (Taghipour and Jalali, 2013; Badr and Taalab,2005) and significantly reduces soil adsorption function and its rate of phosphorus. Meanwhile, humic acid has a certain activation and stimulation effect on soil phosphorus (Liet al., 2011). EDTA is a highly effective chelating agent that can combine with soil metal elements, such as calcium, magnesium and zinc, to form soluble chelates, enhancing the synergy among trace elements and improving the effectiveness of calcium, magnesium and zinc, which are all available to plants. Researches have shown that the application of chelated fertilizer (amino acid, humic acid, EDTA, etc.) to soil can increase crop yield and fertilizer utilization rate (Shao and Xu, 2006). Urrutiaet al.(2013) have shown that humic acid can reduce the fixation of soil phosphorus through the phosphatemetal humic acid complex (PMHA) formed by metal bridge and phosphate, which can effectively increase the absorption of phosphorus and iron by crops.

At present, there are many studies on improving soil physical and chemical properties and soil fertility by applying single chelating agent. However,there is a few studies on the combination of the two chelating agents to improve the rotation of vegetable fields in black soil and correct the symptoms of Chinese cabbage deficiency. Thus, soil incubation experiment and pot experiment was carried out by applying two kinds of chelating agents with different ratios to the vegetable field to investigate the effects of chelating agents on soil calcium and phosphorus availability and cabbage seedling growth, and to find the best chelating agent ratio. Therefore, a theoretical basis was provided for the chelating agent to improve the availability of calcium and phosphorus in soil,which should enhance the resistance of Chinese cabbage and reduce the physiological diseases of cabbage and increase the yield of Chinese cabbage.

Materials and Methods

Experiment materials

The experiment was conducted in May 2018, at the New Fertilizer Laboratory of the College of Resources and Environmental Sciences, Northeast Agricultural University. Experiment materials included lime (CaO),sodium humate and disodium edetate. The experiment crop was Super Xiayang cabbage, the experiment soil samples were taken from the cabbage planting base in Donghuan Village, Acheng District, Harbin City.After the soil samples were naturally air-dried, stones and non-rotted residues of plants were removed, all of which were ground into 2 mm, mixed and stored for later use. The available nitrogen of the experimented soil was 216.47 mg · kg-1, the available phosphorus content was 53.54 mg · kg-1, the available potassium content was 270.29 mg · kg-1and the organic matter content was 31.05 g · kg-1, pH 6.59.

Experiment design

The experiment was carried out including soil incubation and pot experiment. Two kinds of chelating agents were applied: humic acid (F) and EDTA (E),and three levels of 0, 1 and 2 were set, respectively.The experiment was designed as a completely randomized trial with a total of nine treatments, with three replicates per treatment. The treatment numbers were F0E0, F1E0, F2E0, F0E1, F0E2, F1E1, F1E2,F2E1 and F2E2. The treatment applied without humic acid, EDTA and lime was taken as control(CK). According to the previous field test and the experiment on screening the amount of quicklime by potted cabbage, except for CK treatment, other nine treatments were applied with the same amount of lime,675 kg · hm-2. The specific experiment design is shown in Table 1.

Table 1 Experiment design

Soil incubation experiment

The treated soil samples were separately loaded into a one liter jar, 500 g of air-dried soil in each jar, then added distilled water with a maximum soil capacity of 40%. The bottle mouth of jar was sealed with Vaseline and placed in a incubator with light avoidance and constant temperature of 25℃ for 48 days. On the 0th,12th, 24th, 36th and 48th days after the beginning of incubation, about 80 g of soil samples were taken out in each jar, respectively. The soil samples taken out were naturally air-dried, ground and sieved for determination of soil indicators.

Pot experiment

The plastic pot used in the experiment had a hole at the bottom and the drainage and ventilation were in good condition. The specific steps were as the following:put 1.5 kg of air-dried soil sample through 2 mm sieve into the plastic pot, weighed 0.134 g · kg-1urea,0.087 g · kg-1diammonium phosphate, 0.08 g · kg-1potassium chloride (the amounts of N, P and K were N 0.08 g · kg-1dry soil, P2O50.04 g · kg-1dry soil and K2O 0.048 g · kg-1dry soil). Lime, sodium humate,disodium edetate and base fertilizer were separately applied to various treated soil and mixed evenly. The experimented cabbage variety was Super Xiayang,the full and consistent seeds were selected and sown in plastic pots, 30 seeds per pot, evenly distributed.Samples were taken regularly after germination, and finally two plants with the same growth rate were planted in each pot and continued to be cultured. The experiment was started on April 24 and harvested on July 12, 2018, with regular and quantitative watering.

Experiment methods

After measuring the fresh weight and other physical properties of the harvested plants, the plants were killed at 105℃, dried to constant weight at 75℃and weighed the dry weight, and the dry samples were ground and stored in self-sealing plastic bags.Determination of basic physical and chemical properties of soil with reference to analyze of soil; soil pH and conductivity by pH meter and conductivity meter was extracted by water of soil-water ratio 1 : 2.5; soil available phosphorus was extracted by NaHCO3and determined by ammonium paramolybdate colorimetric method; soil available calcium was determined by atomic absorption spectrophotometer after extracted by ammonium acetate; plant samples were digested by concentrated H2SO4-H2O2and calcium and phosphorus were determined by atomic absorption spectrophotometer and ammonium paramolybdate colorimetric method, respectively.

Data analysis

Data were processed by SPSS22.0 software for ANOVA and two-factor analysis, and by Microsoft Excel 2010 software for charting.

Results

Effects of two chelating agents and their interactions on soil and cabbage potted plants

It could be seen from Table 2 that there were differences on soil and Chinese cabbage indicators by applying humic acid, EDTA and both of them. There were significant differences on soil pH and biomass of cabbage and very significant differences on phosphorus accumulation of Chinese cabbage with applying humic acid. With applying EDTA, soil pH, available phosphorus, cabbage biomass and cabbage phosphorus accumulation had significant differences or very significant differences among that of other treatments. Except soil conductivity and soil available calcium content, the interaction between humic acid and EDTA had significant influnce on other indicators among the treatments.

Table 2 Effects of two chelating agents and their interactions on soil and cabbage potted plants

Effects of two chelating agents on soil pH and EC

There were significant differences on soil pH among humid acid treatments and EDTA treatments and very significant differences on soil pH among their interaction treatments. It could be seen from Fig. 1,there were different effects on soil pH of the two chelating agents treatments with different application amounts. In 48th days of incubation, the pH of each treatment was increased from the 0th day to the 12th day after incubation beginning, and gradually decreased after reaching a peak. In the last period of incubation, on the 48th day of incubation, pH of the soil basically tended to be consistent and pH differences among treatments were not significant any more, in which the soil pH of F1E1 treatment was significantly higher than that of other treatments.On the 0th, 12th, 24th and 36th days of soil incubation, soil pH of F1E1 treatment increased by 4.42%,2.40%, 0.45% and 2.30%, respectively, compared with CK treatment, and increased by 4.42%, 1.64%,0.15% and 2.93%, respectively, compared with F0E0 treatment.

Fig. 1 Effects of two chelating agents on soil pH

As could be seen from Fig. 2, there was a significant difference on soil EC values between the two chelating agents with different application amounts. In the 48th days of incubation, the EC value of soil fluctuated during the incubation period, and the minimum EC values all appeared on the 12th day. Except for the 24th day of incubation, EC values were significant different among treatments in other periods. The effects of the combination of humic acid and EDTA treatments on reducing the soil EC value were more effective than those of applied humic acid alone or EDTA treatment. On the 0th, 12th, 24th, 36th and 48th days of soil incubation, F1E1 treatment was reduced by 10.04%, 0.39%, 3.68%, 6.42% and 5.33%,respectively, compared to F0E0 treatment and the F2E2 treatment was reduced by 5.89%, 3.71%, 4.14%,5.50% and 5.09%, respectively, compared to F0E0 treatment.

Fig. 2 Effects of two chelating agents on soil EC

Effects of two chelating agents on soil available calcium and available phosphorus

As could be seen from Fig. 3, except for F1E2 treatment, the soil available calcium of other treatments reached the maximum value on the 24th day. The trend of soil available calcium content of F1E0, F1E1,F2E2 and CK treatments gradually fluctuated during the incubation period. On the 0th day of incubation beginning, the available calcium content of each treatment ranged as F1E2＞F0E0＞F0E1＞F2E2＞F0E2＞F 1E1＞F1E0＞CK＞F2E0＞F2E1, and their differences among treatment were significant. The soil available calcium of F1E2 treatment was significantly higher than that of CK treatment by 15.39%. On the 12th day of incubation, the available calcium content of each treatment changed as F1E1＞F0E1＞F2E0＞F2E1＞F2E2＞F1E0＞F1E2＞F0E0＞F0E2＞CK, in which the differences among treatments were significant,and the soil available calcium of F1E1 treatment was significantly higher than that of CK treatment by 13.11%. On the 24th day of incubation, the available calcium content of each treatment changed as F0E1＞F2E1＞F0E0＞F2E0＞F1E1＞F2E2＞F1E0＞F1E2＞F0 E2＞CK, in which the differences among treatments were significant, and the soil available calcium of F1E1 treatment was significantly higher than that of CK treatment by 6.43%; the soil available calcium of F2E1 treatment was significantly higher than that of CK treatment by 8.76%. On the 48th day of soil incubation, there were no significant differences of available calcium among treatments.

It could be seen from Fig. 4 that the soil available phosphorus content of F1E0 and F0E1 treatments gradually increased from the 0th to the 36th days of incubation, gradually decreased from the 36th to the 48th days, and reached the maximum value on the 36th day. The trend of soil available phosphorus contents of F1E0 and F0E1 treatments gradually increased from the 0th day to the 24th day, gradually decreased from the 24th day to the 48th day, and reached the maximum value on the 24th day. The trend of soil available phosphorus content of other treatments decreased slightly from the 0th day to the 12th day, gradually increased from the 12th day to the 24th day, gradually decreased from the 24th day to the 48th day, and reached the maximum value on the 24th day. On the 24th day of incubation, the soil available phosphorus content of each treatment changed as F1E1＞F1E2＞F0E2＞F2E0＞F0E1＞F1E0＞F2E2＞F0E0＞F2E1, in which the differences of available phosphorus among treatments were significant; available phosphorus of F1E1 treatment was significantly higher than that of F0E0 treatment by 18.12%. On the 36th day of incubation,the soil available phosphorus content of each treatment changed as F2E2＞F2E0＞F1E0＞F0E0＞ F1E1＞F0E1＞F0E2＞F1E2＞F2E1, in which the differences among treatments were significant, F2E2 treatment was significantly higher than F0E0 treatment by 11.46%.On the 48th day of incubation, the soil available phosphorus content decreased in each treatment.

Fig. 3 Effects of two chelating agents on soil available calcium content

Fig. 4 Effects of two chelating agents on soil available phosphorus content

Effects of two chelating agents on biomass of potted Chinese cabbage

There were significant differences on biomass of potted Chinese cabbage among humid acid treatments and EDTA treatments, and very significant differences on biomass of potted Chinese cabbage among their interaction treatments. It could be seen from Table 3 that compared with CK treatment, the biomass of potted Chinese cabbage had significant difference among treatments of two chelating agents with different application amounts. Among them, the biomass of potted Chinese cabbage of F2E2 treatment was significantly higher than that of other treatments,which was 183.43% higher than that of CK treatment.The biomass had no significant difference between that of F2E0 treatment and CK treatment, and the biomass of potted Chinese cabbage of F2E0 treatment was significantly lower than that of other treatments, which was only 7.70% higher than that of CK treatment.There was no significant difference of biomass among F0E0, F1E0, F0E1, F0E2 and F2E1 treatments.

Effects of two chelating agents on accumulation of calcium and phosphorus in potted cabbage

There was no significant difference on the accumulation of calcium in potted cabbage among humid acid alone or EDTA treatment, but there were very significant differences among humid acid and EDTA interaction treatments. It could be seen from Table 4 that compared with CK treatment, calcium accumulation had significant differences among the two chelating agents treatments with different application amounts.The accumulation of calcium in potted cabbage of F2E2 treatment was significantly higher than that of other treatments, which was higher than that of CK treatment by 98.21%. There was no significant difference on calcium accumulation between that of F2E0 treatment and CK treatment, and the calcium accumulation in potted cabbage of F2E0 treatment was significantly lower than that of other treatments, which was only higher than that of CK treatment by 11.67%.

There were significant differences on the accumulation of phosphorus in potted cabbage not only between the two chelating agents treatments with different application amounts, but also among their interaction treatments. It could be seen from Table 4 that the treatments with applying different amounts of the two chelating agents had different effects on phosphorus accumulation in cabbage compared with that of CK treatment. The accumulation of phosphorus in potted cabbage of F2E2 treatment was significantly higher than that of other treatments, which was higher than that of CK treatment by 98.33%. The accumulation of phosphorus in potted cabbage of F0E1 and F0E2 treatments had no significant difference compared with that of CK treatment, which were only 15.35% and 11.07% higher than that of CK treatment,respectively.

Table 4 Spearman correlation analysis among potted biomass, plant indicators and soil indicators

Spearman correlation analysis among various indexes of Chinese cabbage and soil indexes

As could be seen from Table 4, the soil pH showed extremely significant negative correlation with soil EC value, the soil pH showed extremely significant positive correlation with the soil available calcium. There were positive correlations among soil pH, soil available phosphorus, cabbage biomass,cabbage calcium accumulation and cabbage phosphorus accumulation. Soil EC value showed extremely significant positive correlation with soil available calcium. Soil EC value was positively correlated with soil available phosphorus, cabbage biomass and phosphorus accumulation in cabbage, respectively, and was negatively correlated with calcium accumulation in cabbage. Soil available calcium content showed negative correlation with soil available phosphorus content, cabbage biomass, calcium accumulation in cabbage and phosphorus accumulation in cabbage. Soil available phosphorus content was negatively correlated with cabbage biomass and cabbage phosphorus accumulation. Soil available phosphorus content showed extremely significant negative correlation with calcium accumulation in cabbage. Cabbage biomass showed extremely significant positive correlation with calcium accumulation in cabbage and phosphorus accumulation in cabbage.Calcium accumulation in cabbage showed extremely significant positive correlation with phosphorus accumulation in cabbage.

Discussion

In this experiment, there were significant differences on soil pH between humid acid treatments and EDTA treatments and there were very significant differences on soil pH between humid acid and EDTA interaction treatments. pH of CK treatment was significantly lower than that of other applied lime treatments, and pH of F0E0 treatment in each period was lower than that of the treatments with applying the chelating agent. It indicated that during the incubation period pH of the soil could increase by applying the one chelating agent alone, but of which effect was not better than that of applying two chelating agents together. It showed that humid acid and EDTA treatments could regulate soil pH. However, in the later stage of the incubation, pH of each treatment approached to similar, which was due to the fact that soil itself had a strong buffering capacity. On the other hand, it might be that the application of chelating agent formed a relatively stable buffer system with various ions in the soil, thereby regulating and stabilizing pH of the soil. During the incubation period, the EC value of CK treatment was higher than that of the treatments of applying lime, and the EC value of F0E0 treatment was higher than that of the treatments of applying chelating agent. The effect of the combination of humic acid and EDTA treatments on reducing the EC value of soil was more effective than the treatments of applying humic acid or EDTA alone. Research showed that the application of zeolite in soil could reduce the pH and EC values of continuous cropping soil (Weiet al., 2010). Studies showed that the application of humic acid fertilizer on the basis of the application of lime could effectively improve the acidic soil (Liet al., 2017). Liet al. (2015)also thought that the complexing ability of the chelate could effectively inhibit the increase of salt-based ions in the soil, thereby reducing the EC value of salinealkaline soil. Hart and Cornish (2012) also showed that crop yield nonlinearly decreased with the increasing of EC value, which was consistent with the results of this experiment.

In this experiment, there were very significant differences on the soil available phosphorus among EDTA treatments and their interaction treatments.The available calcium content of CK treatment was lower than that of other treatments, because the application of lime effectively supplemented with calcium to soil. For the treatments with chelating agents of applying the same amount of lime, the soil available calcium and phosphorus increased in most cases. From the structure of humic acid, because of its multiple functional groups, the availability of calcium ions in soil could be increased by chelation. There was no significant difference on the soil available phosphorus, which might be that soil nutrient was highly resistant to be transformed, resulting in weak chelation of the chelator. On the other hand, it might be that the amount of chelating agent was too little to result in weak chelation. Wanget al. (2007) showed that the soil available phosphorus content in the soil layer could increase by applying humic acid, and the application amount should reach a certain level to activate a significant effect on the fixed phosphorus in the soil. The soil available phosphorus content of F0E0 treatment was lower than that of CK treatment in each incubation period, which might be due to the increase in soil pH after the application of lime, hydrolysis of aluminum in the soil produced a hydroxy aluminum polymer that adsorbed phosphorus, resulting in a decrease in soil phosphorus availability, which was consistent with Huet al(2017). At the same time,studies showed that humic acid formed a phosphatemetal-humic acid complex (PMHA) by a metal bridge and phosphate. Multiple functional groups in humic acid competed with phosphate ions for adsorption sites in soil colloids, so that it could effectively reduce the fixation of phosphorus in the soil, and the phosphorus and metal elements in the complex could be absorbed by the crop (Urrutiaet al., 2013). In addition, studies showed that the reduction of fixed phosphorus in the soil after application of humic acid might be related to the negative charge of humic acid itself. Because the available phosphorus in the soil was negatively charged and the humic acid had a repelling effect on it.Thereby, the fixation of phosphorus in the soil reduced(Li, 2018).

In this experiment, there were extremely significant differences on the biomass of cabbage, the accumulation of calcium in cabbage and the accumulation of phosphorus in cabbage among the interaction of the two chelating agents. The change of calcium and phosphorus accumulation in Chinese cabbage plants could indirectly reflect the change of chelating agents on soil calcium and phosphorus availability.Compared with CK treatment, cabbage biomass,calcium accumulation and phosphorus accumulation of the treatments applying lime and the two chelating agents increased significantly, which was consistent with the results of Weiet al. (2007), Muet al. (2008)and Caoet al. (2010), whose results indicated that the absorption of calcium and phosphorus of Chinese cabbage could increase by applying chelated fertilizer and there were significant differences on the yield of Chinese cabbage. Studies showed that humic acid could increase the root activity of crops and improve the absorption and utilization of nutrients by crops(Jindoet al., 2012). Liet al. (2017), Xieet al. (2009)and Guoet al. (2010) showed that after applying the soil amendments, pH of acidic soil was adjusted,and the physical and chemical properties of the soil were improved, which were more conductive to soil nutrients release and transformation, so that promoted the growth of crop roots and shoots.

Conclusions

Among the nine ratios treatments of the two chelating agents, cabbage biomass, cabbage calcium and phosphorus accumulation got the maximum value of F2E2 treatment (appied 675 kg · hm-2of lime,300 kg · hm-2of humic acid and 150 kg · hm-2of EDTA)had significant differences with other treatments.Therefore, in this experiment, the chelating agent ratio of F2E2 treatment was optimum.