Introduction

Zeolites are naturally occurring hydrated aluminosilicates comprising about 50 mineral types, including clinoptilolite. Given the natural properties of zeolites, particular attention has been paid in agriculture because they encompass a number of applications (^{Ramesh et al., 2015, 25}). They have a rigid three-dimensional crystal structure with voids and channels of molecular size and a high cation exchange capacity (CEC), arising from the substitution of aluminum (Al) for silicon (Si) in the silicon oxide tetrahedral units that constitute the mineral structure (Figure 1).

Figure 1 (A) Basic zeolite structure. (B) Tetrahedral representation of a natural zeolite. The main building units zeolites are tetrahedral formed by [SiO₄]^4- and [AlO₄]^5- linked together forming oxygen bridges. 

These unique minerals are being used in agricultural production in order to increase water retention and generate more effective fertilizer availability for plants while minimizing nutrient losses (^{Ozbahce et al., 2015, 616}). Ammonia loss and nutrient leaching from fertilizers can be reduced by the application of materials such as clinoptilolite zeolite, which is high in CEC (^{Palanivell et al., 2016, 709}). When zeolites are mixed with fertilizers, they retain nutrients and therefore increase the long-term soil quality by enhancing its absorption readiness. It concerns the most important plant nutrients such as nitrogen, phosphorous, potassium, calcium, magnesium and microelements (^{Orha et al., 2015, 1}).

Furthermore, it has been postulated that zeolite also contributes to sustainable agriculture by preventing the occurrence of environmental problems through increasing N, P, and water-use efficiency in agroecosystems. One way of improving P use efficiency by means of natural zeolite, such as clinoptilolite, would be by increasing soil P availability for plants; zeolite can exchange ammonia to react with phosphate rock substituting calcium in the process and resulting in higher P solubility (^{Shokouhi et al., 2015, 1}).

Zeolites are widely used in agriculture either for plant growing on open field, or as substrate for application under greenhouse conditions (^{Tsintskaladze et al., 2016, 164}). Zeolites are a good source for slow release materials of certain mineral elements required as macro or micro nutrients for plants, and its combination with peat moss and perlite is one of the best cultivation media (^{Eghtedary-Naeini et al., 2016, 523}). It is well known that peatmoss as a substrate can be a suitable organic material for growing plants and improve sustainability in soilless culture (^{Al-Ajlouni et al., 2017, 34}).

There are several reports related to the use of zeolites as substrates for hydroponics. ^{Aghdak et al. (2016, 967)} observed that the incorporation of 25% zeolite in perlite substrate, showed a significant increase in leaf area as well as fresh and dry biomass in pepper plants. Other results also showed that using perlite and zeolite as the growing media produced the highest fruits number and yield. ^{Djedidi et al. (2001, 32)} observed that tomato plants grown in perlite and zeolite at 2:1 ratio, had the best distribution of fruit size; though total soluble solid and the highest fruit dry matter was found in the treatments with perlite as the substrate.

When zeolites are integrated into the soil, they can retain essential nutrients in the root zone, allowing them to be used by plants when required. Consequently, this leads to a more efficient use of fertilizers by reducing their normal application rates, by prolonging their activity, or finally by producing higher yields (^{Leggo, 2015, 2}). During the past 60 years, crop protection and production have relied heavily on synthetic inputs, which damage the environment. In this scenario, synthetic N fertilizer use, combined with other agricultural N inputs (i.e. legumes, crop rotations, and manure applications) to increase productivity over time, has resulted in elevated levels of nitrate in water resources throughout many agricultural regions (^{Masarik et al., 2014, 241}).

Consequently, zeolitic inputs into the soil could relieve environmental pressure such as the increase of N concentration in groundwater recharged by agricultural lands, which has been globally linked to a steady increase of nitrate loading to surface waters; so, the application of zeolites can both prevent nitrogen leaching and improve soils physical properties (^{Afrous and Goudarzi, 2015, 56}). For example, the loading of N and P from primarily Midwestern states in the United States, has been linked to the increase of hypoxic zones in the Gulf of Mexico (^{Rabalais et al., 2002, 236}).

The use of synthetic agrochemicals for plant nutrition or for disease management, pests, weeds, etc., has become a common practice around the world. However, environmental pollution and ecological issues force us to find new strategies, such as the use of bioorganic agrochemicals or the controlled release of fertilizers and pesticides, including a reduction in the amount of active ingredients (^{De Smedt et al., 2015, 1356}). Lately, the interest in restructuring the agricultural supply chain for the development of sustainable agriculture has increased (^{Priya and Vivek, 2016, 136}), and hence the use of natural products such as zeolites to improve physical and chemical properties of the soil (^{Moraetis et al., 2016, 13274}).

Based on the above-mentioned rationale, we hypothesized that by using Mexican zeolite-clinoptilolite (ZC) as substrate, we would increase biomass production and growth of S. lycopersicum plants. Therefore, the objective of this study was to analyze the effect of ZC on plant growth and biomass production when incorporated to the substrate at different rates.

Materials and methods

Results

Experiment 1. Physicochemical properties of substrate blends

The chemical tests of the natural ZC employed in this study (Table 1), point out that this type of aluminosilicate is rich in potassium and calcium, and that it also harbors other macro and micronutrients.

Table 1 Chemical properties of natural zeolite-clinoptilolite (ZC). 

Chemical composition (mg kg-1)										Texture %			M.O.%	pH
N	P	K	Ca	Mg	Fe	Cu	Zn	Mn	B	Sand	Silt	Clay
17.2	1.43	17360	2641	385	3.8	0.31	0.64	6.77	0.75	52.2	34	13.8	0.4	8.75

Regarding the substrates physical characterization (Figure 2), treatments containing 20% and 30% ZC, presented a statistical increase (p≤0.05) of total porosity (Figure 2A), particularly T4 (16.6%) and T5 (8.47%), compared to control treatment (T1). All substrates supplemented with ZC promoted greater water holding capacity (WHC), since T4 and T5 exhibited a superior volume of water retained (35.33% and 33.71%, respectively), followed by T3 (25.85%) (Figure 2B). The lesser WHC was attained by T1 (9.34%) without ZC added. Concerning to bulk density (Figure 2C), this variable was increased significantly (p<0.05) by T4 (0.21 Mg m^-3) and T5 (0.25 Mg m^-3), compared to T1 (0.08 Mg m^-3). The addition of ZC to the substrate also improved particle density (Figure 2D), since T4 and T5 reported 0.37 Mg m^-3 and 0.43 Mg m^-3 respectively, compared to T1 (0.13 Mg m^-3).

Figure 2 Physical properties of substrate mixtures tested on S. lycopersicum seedlings. Error bars represent the standard error of the mean (n=3); columns within the same graph with different letter are statistically different according to Tukey test (α=0.05). An assay was set to test and compare the physical properties of three substrates: peat moss (pm), perlite (per) and zeolite (zeo), and their mixtures (pm:per:zeo) at different proportions. T1 (control) = 100:0:0; T2 = 70:30:0; T3 = 70:20:10; T4 = 70:10:20 and T5 = 70:0:30 (v/v). 

Experiment 2. Effect of zeolite-clinoptilolite (ZC) on Solanum lycopersicum seedlings growth

Tomato plants grown on ZC substrates exhibited higher growth (Figure 3), compared to control plants (100% peat moss). Among ZC-containing substrates, peat moss mixed with ZC at a 70:30 ratio (T4), produced higher plants (24.2%, Figure 3A, and Figure 4A); T5 produced greater number of leaves (92.0%, Figure 3B), root length (63.23%, Figure 3C, and Figure 4B); also were increased stem diameter (28.5%, Figure 3D), as well dry weight of aerial part (62.5%), root dry biomass (208.9%) and leaf area (64.5%) than the control plants (Table 2).

Figure 3 Response of S. lycopersicum plants to natural zeolite incorporated to substrates mixtures. T1 = 100:0:0; T2 = 70:30:0; T3 = 70:20:10; T4 = 70:10:20 and T5 = 70:0:30 (peat moss: perlite: zeolite (v:v) respectively). Error bars represent the standard error of the mean (n=12); columns within the same graph with different letter are statistically different according to Tukey test (α=0.05). 

Figure 4 (A) Aerial biomass and root length (B) of tomato seedlings grown on substrates containing zeolite clinoptilolite. 

Nevertheless, pm: per: zeo at 70:20:10 (T3) and 70:10:20 (T4) ratios were the substrates that produced plants with superior root growth (Table 2). On the contrary, relative chlorophyll content of leaves (SPAD index), was the only parameter which showed no significant differences (p>0.05) among plants grown on any of the substrates tested.

Table 2 Traits of tomato plants grown on substrates containing different proportions of peat moss, perlite and zeolite-clinoptilolite. 

Treatments
Traits	T1 100:0:0	T2 70:30:0	T3 70:20:10	T4 70:10:20	T5 70:0:30	CV (%)	p>F
Leaf area/plant (cm2)	478.80±42b	706.41±86a	701.14±88a	695.82±34a	787.89±46a	22.2	0.020*
Aerial part dry weight (g)	2.49±0.34a	3.51±0.33ab	3.66±0.37ab	3.52±0.20ab	3.94±0.27b	21.4	0.025*
Root dry biomass (g)	0.67±0.07a	1.87±0.44ab	2.47±0.43b	2.34±0.62b	2.07±0.20ab	50.0	0.025*
Chlorophyll content‡	39.12±2.0a	40.26±1.0a	40.70±2.0a	38.78±1.0a	41.25±1.0a	11.4	0.866ns

Means (n=12); ± SE in the same row with different letters, are statistically different (Tukey’s, p≤0.05); * = Significantly different; ‡Spad value; ns= No significance between treatments; CV= Variation coefficient.

Discussion

Substrates containing ZC significantly increased the water holding capacity compared to substrates without ZC added, this means water in the treated substrate can be more readily adsorbed by crop plants. Thus, ZC has special properties that can be potentially applied to increase water use efficiency in agriculture. Several experiments carried out in pots at the seedling stage agree with our results. The results of ^{Yılmaz et al. (2014, 2771)} point out that mixtures of peat moss; peat moss and zeolite (20 % and 100% v / v), significantly promoted better germination, plant height, stem diameter, fresh weight and nutrient content in dry biomass of cucumber seedlings. The outcome of ^{Manolov et al. (2006, 487)} stated that zeolite mixed with perlite (1:1 v/v), stimulated greater dry biomass, root length and leaf area of tomato and cucumber seedlings after 25 days of evaluation. Likewise ^{Gul et al. (2005, 464)} detected a growth increase of N and K content in lettuce plants, and a reduction of K leaching on substrates containing zeolite at 50 and 70%. ^{Bernardi et al. (2010, 435)} documented the highest increase in growth and dry biomass production of lettuce, tomato and rice plants, with the incorporation of 20, 40, 80 and 160 grams per 3 kilo of perlite substrate added.

It had been demonstrated that zeolite mixed with other substrates can hold macro and micro nutrients, such as K and N from the chemical fertilization, therefore reducing its loss by percolation, favoring its availability, and functioning as a slow release fertilizer in grain crops such as Triticum aestivum and Avena sativa (^{Orha et al., 2015, 4}). This fact is of great significance for tomato and other vegetable crops, since K has a very important function in pH stability, enzymatic activation, photosynthesis, protein synthesis, cellular elongation and stomatal aperture (^{Najafi-Ghiri, 2014, 33}; ^{Orha et al., 2015, 4}).

Results reported by ^{Xiubin and Zhanbin (2001, 45)} show that soil treated with zeolite, compared to normal soil, increase infiltration by 7-30% on gentle slope land and more than 50% on steep slope land. In addition, soil amended with zeolite increased soil moisture by 0.4-1.8% in extreme drought condition and 5-15% in general situation. Consequently zeolites improves water use efficiency, as well the effectiveness of fertilizers such as nitrogen, phosphorous and organic manure incorporated to the soil (^{Ramesh et al., 2011, 228}).

In the present study the growth enhancement showed by S. lycopersicum plants cultured in the presence of ZC incorporated into the substrate, agrees with several reports related to different crop plants grown with this aluminosilicate as substrate. For example, ^{Kavoosi (2007, 69)} reported that zeolite applied to substrates at the rate of 8, 16 and 24 t ha^-1 increased the yield of rice plants by 19%, and all levels of zeolite with N added, statistically increased grain yield; zeolite also increased significantly soil-available K and its uptake by rice straw.

Incorporation of zeolite to substrates enhances N uptake by plants (^{Lija et al., 2014, 284}), and since it is a major plant nutrient involved in protein and chlorophyll chemical structures, as well in many more important molecules, its availability impacts crop yield (^{Malekian et al., 2011, 970}). Zeolite also acts as a mineral fertilizer, because it is a natural source of N, K, Ca, Mg, Fe and other minerals. In the present study the high K content might have been assimilated by S. lycopersicum plants and then positively impacted photosynthesis by improving the water dynamics of plants, namely stomata conductance and transpiration (^{Abdi et al., 2006, 388}).

An improved yield effect due to zeolite addition also was found in Brassica napus plants by ^{Shahsavari et al. (2014, 1813)}, since the greatest proportions of all studied traits were attained by applying Z₂Zn₂ (15 t ha^-1 zeolite and 0.1% Zn sulfate). The rates of grain yield, biological yield, and harvest index improved by 43.8 %, 73.9 %, and 30.0%, respectively, by the pooled application of Z and Zn.

In a similar way, ^{Yilmaz et al. (2014, 2771)} described that soil-applied zeolite had a positive effect on nutrients contents of Cucumis sativus plants. Likewise, ^{Malekian et al. (2011, 973)} described that grain yield, grain N content, dry matter, and N uptake of Zea mays were significantly greater in soil amended with 60 g kg^-1 of zeolite-clinoptilolite. ^{Najafinezhad et al. (2014, 234)} reported that for corn and sorghum crops, treatments with zeolite had the highest forage yields and the lowest cadmium concentrations in the tissue. Using 4.5 t ha^-1 residues, significantly increased relative water content and decreased leaf malondialdehyde and proline; consequently, they recommend planting corn using 5 t ha^-1 zeolite and 4.5 t ha^-1 residues in a double-cropping system.

In consideration of our results, we conclude that the zeolite-clinoptilolite, which was obtained from a Mexican natural land deposit, promoted plant growth and biomass accumulation of S. lycopersicum seedlings, by means of substrate amendment, since the ionic mobility of major cations is greatly increased in the zeolitic substrates used for greenhouses or shade houses. However, our results have to be validated under open field conditions or in protected agriculture.