Effects of humic acid and Ca(NO₃)₂ on nutrient contents in pepper (Capsicum annuum) seedling under salt stress

ABSTRACT

This study was carried out in order to determine the effects of calcium nitrate (Ca(NO₃)₂) and humic acid (HA) applications on nutrient uptake of pepper seedling under salt stress in a plant growth room. Before sowing Demre variety of pepper seeds, 60 mM NaCI was added to each 300 cm³ pot. The experiment was ended at eighth week after the sowing. Applications of HA significantly affected K, Ca, Fe, Mn, Zn (p < .01) and P (p < .05) contents. Also, Ca(NO₃)₂ applications did not show a significant effect on Fe and Cu contents of pepper seedling. However, Ca(NO₃)₂ applications significantly affected N and P contents (p <.05) and K, Ca, Mn, and Zn contents (p < .01) of pepper seedling under salt stress.

KEYWORDS:

• Pepper seedling
• salt stress
• humic acid
• calcium nitrate
• nutrients

Introduction

The total salt concentration of the nutrient solution has an important effect on the commercial yield (Sonneveld et al. 1999). Generally, plant growth and productivity is restricted due to salinity in arid and semi-arid regions. The effects of salinity on plants are complex (Savvas & Lenz 1994). The injurious effects of salinity are associated with deficits, ionic imbalance, mineral nutrition, stomata behaviors, photosynthetic efficiency, and carbon allocation and utilization. Several researchers (Bohmert et al. 1995; Kuznetsov & Shevyakova 1997) reported that high concentrations of salts cause water deficiency, ion imbalance, ion toxicity, malnutrition or a combination of any of these adverse factors. Therefore, the water potential can reduce and ion distribution can affect within plants (Carvajal et al. 1999). Grattan and Grieve (1999) reported that nutrient imbalances can occur in salt-stressed plants in various ways. Imbalances may be caused by the effect of salinity on nutrient availability, competitive uptakes transport or partitioning within the plant. Drastic changes in ion concentrations of issues cause molecular damage, growth arrest and even death (Zhu 2001). The interactive nature affecting nutrient availability, uptake and distribution is highly complex in the absence of other stress conditions (Marschner 1995). Humic substances (humic and fulvic acids) constitute 65–70% of the organic matter in soils. However, the mechanism of humic acids activity in promoting plant growth is not completely known; some researchers (Russo & Berlyn 1990) explained that humic substances increase cell membrane permeability, oxygen and phosphate uptake, respiration and root cell elongation. Many researchers reported that humic acid application increased the plant growth and the nutrient uptake by plants. They also reported that humic acid was particularly important for the availability and transport of micro nutrients (Aydın et al. 1999; Dursun et al. 2002). Mohamed (2012) studied on the effects of humic acid and calcium forms on dry weight and nutrient uptake of maize plant under the saline condition. He found that slats caused plant death at no humic acid application, but there was no plant death in humic applications in all of the salt types.

Calcium has an important role in several physiological processes in plants, such as ion transport, translocation of carbohydrates, protein and their storage during seed formation and other enzymatic activities. It has been reported that calcium inhibits Na⁺ uptake and thereby reduces its adverse effect on seed germination (Nayyar 2003; Bonilla et al. 2004) and increases plant growth (Tobe et al. 2001; Munns 2002). van Goor (1968) reported that the main role of calcium in plant growth and nutrient uptake is increasing leakage of low molecular-weight solutes from cells of calcium-deficient tissue. It was also reported that deficiency of calcium in plant tissue caused disintegration of membrane structures and loss of cell formation. It was reported that sodium ions may compete with Ca ions for membrane-binding sites. Therefore, it was thought that high concentrations of Ca can protect the cell membrane from the adverse effects of salinity (Busch 1995; Kaya et al. 2002). The positive effect of calcium application on improvement of imbalance in nutrient uptake by plants under salt stress has been demonstrated by the some studies (Murillo-Amador et al. 2006; Jaleel et al. 2007; Arshi et al. 2015). Salinity injures cell membranes and increases solute leakage. It was reported that these adverse effects are prevented by Ca (Crammer et al. 1985). The addition of Ca to nutrient solution partly decreases the suppression of root growth by salinity (Kent & Lauchli 1985; Shekofteh 2015).

The objective of this study was to determine the effect of different levels of calcium and humic acid applications on nutrient contents of pepper seedling under salt stress.

Materials and methods

In this study, 1:1 ratio of garden soil:sand mixture was used as a plant growth medium. Some properties of the growth medium were determined using standard soil analysis methods (Kacar 1994). The growth medium used in the study had a sandy loamy texture, non-saline, slightly alkaline, low in organic matter and insufficient in phosphorus content (Table 1).

Table 1. Some properties of the growth media.

Total salinity (%)	0.02	P (mg kg^−1)	3.78
pH (1:2.5)	8.12	K (cmol kg^−1)	0.43
Organic matter (%)	0.75	Ca (cmol kg^−1)	28.0

Experimental design

After filling each 300 cm³ pot without drainage holes with the soil:sand mixture, 480 pots were autoclaved. The reason of using pots without drainage holes in the experiment was to supply desired salt conditions in the growth medium with preventing drainage and salt leaching by the irrigation water. Also, the desired moisture content was supplied with avoiding excess irrigation. Four doses of humic acid (HA₀:0, HA₁:1000, HA₂:2000, HA₃:3000 mg kg⁻¹) and Ca(NO₃)₂ (CaN₀:0, CaN₁:50, CaN₂:100, CaN₃:150 mg kg⁻¹) treatments were applied into pots with three replications. Each replication was formed from 10 pots. As a basic fertilizer treatment, 90 mg P₂O₅ kg⁻¹, 180 mg K₂O kg⁻¹ and 250 mg N kg⁻¹ were applied into each pot using the triple superphosphate (TSP), K₂SO₄ and (NH₄)₂SO₄ fertilizers, respectively. To increase salt concentration of the growth medium, 60 mM NaCl was also added into each 300 cm³ pot. The experimental study was carried out in a plant growth room of Horticultural Department in Yuzuncu Yıl University under controlled conditions. Demre pepper variety was used as a plant material. Three pepper seeds were sown in each pot and then the seedling were thinned to one. The pots were placed in a growth chamber at 22 ± 1°C with 12 fluorescent illuminations with 8000 lux light intensity and the seedlings were irrigated with distilled water. The experiment was ended at the eighth week of the sowing.

Nutrient analysis

The nutrient contents of the seedlings were analyzed in dried and ground plant samples according to following methods reported by Kacar and Inal (2008). The N content was determined by the Kjeldahl method, the P level was analyzed by the spectrophotometric method, and K, Ca, Mg, Fe, Mn, Zn, and Cu levels were determined using an atomic absorption spectrophotometer.

Statistical analysis

Variance analyses of the experimental data were accomplished in a completely randomized block design with two treatments (Ca(NO₃)₂ and humic acid) using SAS (1988) statistic program and significantly different means numbered according to Fisher’s Least Significant Difference (LSD) test.

Results

The variance analyses of results and the effects of HA and Ca(NO₃)₂ treatments on plant nutrient contents are given in Tables 2 and 3, respectively. According to the variance analyses, HA applications significantly influenced macro- and micro-nutrient contents of pepper seedlings, except N content. Similarly, CaN applications showed significant effect on macro- and micro-nutrient contents of pepper seedlings, except Fe content (Table 2). The effects of HAxCaN interaction on macro- and micro-nutrient contents, except N and Fe, were found as significant statistically.

Table 2. Variance analyses of the results for nutrient contents of pepper seedlings.

Variation source	DF	Nitrogen (N)		Phosphorus (P)		Potassium (K)		Calcium (Ca)
		M.S.	F value	M.S.	F value	M.S.	F value	M.S.	F value
Humic acid (HA)	3	0.050	0.43^ns	421,323	3.57*	0.135	5.53**	0.434	7.05**
Ca(NO3)2 (CaN)	3	0.389	3.39*	354,478	3.00*	0.706	28.88**	1.736	28.18**
HA × CAN	9	0.232	2.02^ns	369,890	3.12**	0.105	4.30**	0.288	4.67**
Variation source	DF	Iron (Fe)		Manganese (Mn)		Zinc (Zn)		Cupper (Cu)
		M.S.	F value	M.S.	F value	M.S.	F value	M.S.	F value
Humic acid (HA)	3	18,562	9.24**	487.07	7.81**	62.63	15.62**	11.88	3.09*
Ca(NO3)2 (CaN)	3	3456	1.72^ns	344.29	5.52**	27.00	6.73**	11.99	3.12*
HA × CaN	9	1565	0.78^ns	79.98	1.28^ns	11.20	2.79*	12.79	3.32*

*Significant at .05;

**Significant at .01;

^nsNot significant.

Table 3. Effects of humic acid and Ca(NO₃)₂ treatments on nutrient contents in pepper seedlings.

	Humic acid (mg kg^−1)
		Control	1000	2000	4000	Mean
Nitrogen (N, %)
Ca(NO3)2 (mg kg^−1)	Control	3.661	3.858	4.080	3.749	3.837 B*
	50	3.949	4.010	4.430	4.320	4.177 A
	100	4.262	4.075	4.463	4.075	4.219 A
	150	4.622	4.295	3.781	4.059	4.189 A
	Mean	4.123	4.059	4.189	4.051
Phosphorus (P, mg kg^−1)
Ca(NO3)2 (mg kg^−1)	Control	3541 b**	3452 b	4117 a	3758 b	3717 AB
	50	3601 b	3604 b	3044 b	3740 b	3497 B
	100	3639 b	3652 b	3763 b	4360 a	3854 A
	150	3452 b	3649 b	4533 a	3958 ab	3898 A
	Mean	3558 C	3589 BC	3864 AB	3954 A
Potassium (K, %)
Ca(NO3)2 (mg kg^−1)	Control	1.257 b	1.267 b	1.269 b	1.278 b	1.268 B
	50	1.187 b	1.219 b	1.202 b	1.185 b	1.199 B
	100	1.314 b	1.190 b	1.181 b	1.248 b	1.233 B
	150	2.184 a	1.324 b	1.499 b	1.853 a	1.715 A
	Mean	1.485 A	1.250 B	1.288 B	1.391 AB
Calcium (Ca, %)
Ca(NO3)2 (mg kg^−1)	Control	1.089 f	1.240 def	1.203 def	1.165 ef	1.174 C
	50	1.592 b–f	1.532 b–f	1.334 c–f	1.376c–f	1.459 B
	100	1.704 b–e	1.768 bcd	1.463 b–f	1.670 b–c	1.651 B
	150	2.980 a	1.837 bc	1.525 b–f	1.978 b	2.080 A
	Mean	1.841 A	1.594 AB	1.381 B	1.547 B
Iron (Fe, mg kg^−1)
Ca(NO3)2 (mg kg^−1)	Control	212	208	137	221	195
	50	187	168	139	232	181
	100	213	147	126	216	175
	150	297	180	187	290	239
	Mean	227 AB	176 BC	147 C	240 A
Manganese (Mn, mg kg^−1)
Ca(NO3)2 (mg kg^−1)	Control	101	99	103	113	104 A
	50	88	92	84	105	92 B
	100	94	86	94	105	95 B
	150	84	88	101	98	93 B
	Mean	92 B	91 B	95 B	105 A
Copper (Cu, mg kg^−1)
Ca(NO3)2 (mg kg^−1)	Control	11.16 bc	11.98 bc	10.27 bc	10.08 bc	10.90
	50	9.03 c	10.26 bc	10.70 bc	10.30 bc	10.07
	100	12.57 b	8.95 c	10.06 bc	11.53 bc	10.78
	150	12.01 bc	9.89 bc	10.23 bc	17.65 a	12.45
	Mean	11.19	10.27	10.32	12.39
Zinc (Zn, mg kg^−1)
Ca(NO3)2 (mg kg^−1)	Control	11.16 b–e	11.57 b–e	15.55 a	12.64 abc	12.73 A
	50	9.33 cde	10.79 b–e	12.27 a–e	10.36 b–e	10.69 AB
	100	8.79 de	10.87 b–e	12.34 a–d	13.25 ab	11.31 AB
	150	2.50 f	8.74 e	12.14 a–e	13.07 ab	9.11 B
	Mean	7.94 C	10.49 B	13.08 A	12. 33B

*,**Means followed by different capital letters, in columns, and followed by different small letters, in lines, differ statistically at .01.

Mean N content values in HA₂ (4.189%) and HA₀ (4.123%) were higher than mean N content values in HA₁ (4.059%) and HA₃ (4.051%) (Table 3). Increasing CaN doses increased N content significantly (p < .01). While the highest mean N content (4.189%) was determined in CaN₃, the lowest mean nitrogen content (3.837%) was in the CaN₀ treatment.

Humic acid application significantly (p < .05) increased P content. While the highest mean P content (3954 mg kg⁻¹) was determined in HA₃ treatment, the lowest mean P content (3558 mg kg⁻¹) was in HA₀ treatment. The highest mean P content (3898 mg kg⁻¹) determined in CaN₃ treatment was in the same range with mean P content (3854 mg kg⁻¹) in CaN₂ treatment according to the LSD test (Table 3). The lowest mean P content as 3497 mg kg⁻¹ in CaN₁ treatment. The highest P content (4533 mg kg⁻¹) and the lowest P content (3452 mg kg⁻¹) values were determined in HA₀ × CaN_3, HA₁ × CaN₀ and HA₂ × CaN₂ interactions, respectively (Table 3).

The mean K contents were significantly (p < .01) decreased by HA treatments. The highest and the lowest mean K contents were determined in HA₀ (1.485%) and HA₁ (1.250%), respectively (Table 3). Mean K (1.391%) in HA₃ was in the same group with mean K (1.485%) in HA₀ statistically. Increasing Ca(NO₃)₂ doses, except CaN₃ treatments, significantly (p < .01) decreased the mean K contents. Mean K content values ranged between 1.199% in CaN₁ and 1.715% in CaN₃. Mean values of K content in CaN_0, CaN₁ and CaN₂ were in the same group statistically. The highest K content (1.853%) and the lowest K content (1.181%) were determined in HA₂ × CaN₂ and HA₃ × CaN₃ interactions, respectively.

Humic acid treatments significantly (p < .01) decreased Ca content. The highest and the lowest mean Ca content values were 1.841% in HA₀ and 1.381% in HA₂, respectively (Table 3). Increasing CaN doses increased Ca content significantly (p < .01). While the highest mean Ca content (2.080%) was determined in CaN₃, the lowest mean Ca content (1.174%) was in the CaN₀ treatment. HA₀ × CaN₀ and HA₀ × CaN₃ interactions gave the highest (2.980%) and the lowest (1.089%) Ca content values, respectively (Table 3).

Humic acid treatments, except HA₃ treatment, significantly (p < .01) decreased Fe content. The highest and the lowest mean Fe contents were 240 mg kg⁻¹ in HA₃ and 147 mg kg⁻¹ in HA₂, respectively (Table 3). While the highest and the lowest mean Fe contents were obtained 239 mg kg⁻¹ in CaN₃ treatment and 175 mg kg⁻¹ in CaN₂ treatment.

The HA₃ treatment significantly increased mean Mn content. The highest and the lowest mean Mn content values were 105 mg kg⁻¹ in HA₃ and 91 mg kg⁻¹ HA₁, respectively (Table 3). HA₀, HA₁ and HA₂ treatments were in the same group statistically according to the LSD test. Increasing Ca(NO₃)₂ doses significantly (p < .01) decreased the mean Mn content. While the highest mean Mn content (104 mg kg⁻¹) was determined in CaN₀, the lowest mean Mn content (92 mg kg⁻¹) was in the CaN₁ treatment. On the other hand, while HA₃ × CaN₀ interaction gave the highest Mn content (113 mg kg⁻¹), HA₀ × CaN₃ and HA₂ × CaN₁ interactions gave the lowest Mn content (84 mg kg⁻¹).

Humic acid treatments significantly (p < .01) increased Zn content. The highest and the lowest mean Zn contents were 12.33 mg kg⁻¹ in HA₃ and 7.94 mg kg⁻¹ in HA₀, respectively (Table 3). Increasing CaN doses decreased Zn content significantly. Mean Zn content values varied between the highest (12.73 mg kg⁻¹) in CaN₀ and the lowest (9.11 mg kg⁻¹) in CaN₃. HA₂ × CaN₀ and HA₀ × CaN₃ interactions gave the highest (15.55 mg kg⁻¹) and the lowest (2.50 mg kg⁻¹) Zn content values, respectively.

Mean Cu content values in HA₃ (12.39 mg kg⁻¹) were higher than mean Cu content values in HA₀ (11.19 mg kg⁻¹), HA₁ (10.27 mg kg⁻¹) and HA₂ (10.32 mg kg⁻¹) (Table 3). While the highest mean Cu content (12.45 mg kg⁻¹) was obtained in CaN₃ treatment, the lowest mean Cu content (10.07 mg kg⁻¹) was in CaN₁ treatment.

Discussion

According to the results, it can be concluded that increasing doses of humic acid had positive effects on nutrient contents, except nitrogen, potassium and calcium contents, of pepper seedling (Table 3). It can be thought that increases in plant growth by the humic acids treatments caused a dilution effect on nutrient concentration of pepper seedling. Therefore, nitrogen, potassium and calcium contents decreased compared with the control (Table 3). HA treatments significantly increased zinc content and slightly increased phosphorus and manganese, which were in the same group with the control (HA₀) treatment (Table 3). Similarly, HA₂ treatments significantly increased zinc and phosphorus contents. Positive effects of HA₃ treatments were found significant for phosphorus, manganese and zinc. Ameliorative effects of humic acid on nutrient content under saline soil conditions were reported by most researchers (Türkmen et al. 2005; Aşık et al. 2009). Mohamed (2012) reported that humic acid application with calcium increased N, P, K, Fe and Zn uptake of corn plant under salt stress.

Ca(NO₃)₂ treatments generally increased nutrient content compared with the control except manganese and zinc (Table 3). Increasing Ca(NO₃)₂ doses decreased seedling growth criteria, such as shoot dry weight, root dry weight and stem neck diameter (Gülser et al. 2010). Therefore, levels of nutrient contents were found less in control treatment (HA₀) than in the other treatments. Similarly, Türkmen et al. (2004) reported that Ca(NO₃)₂ treatments increased macro- and micro-nutrient contents in tomato under saline conditions.

It is noticed that 4000 mg kg⁻¹ HA and 100 mg kg⁻¹ and 150 mg kg⁻¹ Ca(NO₃)₂ treatment had a negative effect on seedling growth criteria of pepper seedling (Table 3). It was possible that the nutrient uptake level was less in these treatments than in the other treatments. As a result, it can be suggested that 1000 and 2000 mg kg⁻¹ HA and 50 mg kg⁻¹ Ca(NO₃)₂ doses had positive effect on nutrition of pepper seedling under salt stress.

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes on contributors

Ferit Sönmez has obtained PhD in the subject of soil microbiology and plant nutrition interactions. Presently, he is an academic staff and serving as an Assistant Professor in the Department of Seed Science and Technology, Izzet Baysal University, Bolu-Turkey. He has been completed eight research projects in soil science and plant nutrition. He has published 32 research papers in the international journals and proceeding books. He is involved in researches in the area of soil pollution, plant nutrition, organic fertilizer, plant growth promoting rhyzobacteria, soil conditioners.

Füsun Gülser has obtained PhD in the subject of soil microbiology and plant nutrition. Presently, she is an academic staff and serving as a Professor in the Department of Seed Science and Plant Nutrition, Yüzüncü Yil University, Van-Turkey. She has been completed more than 15 research projects in soil science and plant nutrition. She has published 65 research papers in the international journals and congress proceeding books. Presently, she is involved in researches in the area of drought stress, plant enzymes, soil pollution, macro and micro nutrients, soil conditioners, organic fertilizer.

Additional information

Funding

Authors are grateful to the Yuzuncu Yil University for the financial support of this study as a scientific research project [2006 ZF-B64].