Introduction

Recent studies on the effects of salinity on agricultural crops, show the importance of this problem in world agriculture. Therefore, alternatives have been sought to mitigate the effect of salinity and one of these is the use of bio-stimulants of natural origin, which have been rising to the extent that they demonstrate that they are able to minimize abiotic stresses (^{Munns 2002}; ^{Chinnusamy and Zhu, 2003}; ^{Terry and Leyva, 2006}).

The growing interest of consumers for natural products stimulate the market of aromatic plants, making this a viable option for the agricultural sector, the possibility of exporting fresh or processed extracts, essences and oils used in culinary industries , cosmetic and pharmaceutical (^{Biasutti and Galiñares, 2005}; ^{Terry and Leyva 2006}; ^{Méndez, 2008}). Basil (Ocimum basilicum L.) is an important aromatic species for its use in the food industry as a flavoring and seasoning; in pharmacy as a stimulant, antispasmodic and perfume industry for flavoring cosmetics and fine perfumery (^{Cussaianoviich, 2001}; ^{Klimánková et al., 2008}; ^{Calderín et al., 2012}.). There are no reports of the effects of bio-stimulants as mitigating salinity in growth and development of basil, so the objective of this study was to determine the effect of a natural bio-stimulant (humate of vermicompost) as attenuating of salinity stress on growth and development of two Basil varieties, one tolerant and one sensitive to salinity, in order to elucidate the possible differential response of both varieties to the application of humates.

Materials and methods

Study site. The experiment was conducted in a greenhouse-like structure covered with a black plastic mesh with shadow of 40% in the Center for Biological Research of the Northwest, S.C. (CIBNOR^®), located north to the city of La Paz, Baja California Sur, Mexico and located at the geographic coordinates 24° 08' 10.03 "north latitude and 110° 25' 35.31" west longitude at 7 masl.

Genetic material. Napoletano and Sweet Genovese seed varieties, selected as tolerant and sensitive to salinity in previous experiments performed in germination, emergence and early vegetative stages development according to ^{ISTA (1999)} were used.

Experimental design and treatments. The experiment was conducted using a completely randomized design with a tri-factorial arrangement 2 x 3 x 2, where the factor A were two varieties of basil, a tolerant and sensitive to salinity (Napoletano and Sweet Genovese, respectively), factor B were three concentrations of NaCl (0, 50 and 100 mM) and factor C were two humate vermicompost concentrations (0 and 1/60), with six replications. The seeds of both varieties were disinfected by dipping for 5 min in a solution of calcium hypochlorite, containing 5% active chlorine. The seeds were washed with distilled water and embedded in distilled water (control) or in humates of vermicompost dilutions (1/60 v/v) for 24 hr. analysis of variance and multiple comparisons of means (Tukey, p = 0.05) were performed. Analyses were performed using the statistical program Statistica^® (^{StatSoft Inc2011}) for Windows.

Humate composition. Humates are considered plant bio-stimulator and nutrients carrier (Ca, Mg, Na, P₂O₅, K, N), free amino acids, polysaccharides, carbohydrates, inorganic elements, humified substances, beneficial microorganisms, plant hormones and soluble humus, whose composition by chemical fractions correspond to a pH of 8.7, 53.4% C, 4.85% H, 35.6% O, 3.05% N, 0.72% S, a H/C ratio of 0.08, a ratio 0/C of 0.62, a C/N ratio of 18.4, 4.82 humic acids and 7.17 fulvic acids in E4/E6 ratio of its optical coefficient.

Experimental management. The seeds were sown in trays or 200 cavities polystyrene trays, which contained sogemix PM^MR as substrate. To maintain moisture, daily irrigations were applied in order to achieve a homogeneous seedling emergence. The transplant was performed when plants had an average height of 15 cm in pots of about 1 kg, same containing as commercial substrate sogemix PM^®. In each pot one plant was placed in order to ensure successful transplantation. Once transplanted, it began with the daily application of irrigation, using potable water, which contained a nutrient solution prepared according to ^{Samperio (1997)}. After a week of transplantation, began with the application of saline treatments of 0, 50 and 100 mM NaCl and foliar application of the corresponding dilution of humates vermicompost (1/60 v/v), using distilled water as control.

Each replication was represented by a pot containing a plant each, with 36 pots per variety for a total of 72 pots. During the second week began with gradual application of saline treatment, in order to avoid an osmotic shock in seedlings, according to the methodology proposed by ^{Murillo-Amador et al., (2007)}. The amount applied in each irrigation was 500 mL, achieving that solution applied drained through the pot holes in order to prevent accumulation of salts in the substrate. The pH of the solution with saline treatments and nutrients was adjusted to 6.5 by adding KOH.

Morphometric variables. At 45 days after the application of saline treatment, plants were transferred to the laboratory proceeding to separate root, stem and leaves and measured, stem length (cm), root length (cm), fresh and dry (g) biomass of root, stem, leaf and leaf area (cm²), the latter was determined by a leaf area integrator (Li-Cor^®, model-LI-3000A, PAM 1701 series). An analytical balance (Mettler Toledo, model AG204) was used to determine fresh and dry weight of biomass. For dry biomass at all stages, corresponding to leaves, stems or roots were placed in paper bags and then in a drying oven (Shel-Lab^®, model FX-5, series-1000203) at a temperature of 80 °C until complete dehydration (about 72 h). Then weighed on analytical balance (Mettler Toledo AG204) expressing the weight in grams of dry vegetable matter.

Results and discussion

Morphometric variables. For stem length (LT) significant differences between varieties were found (F_1,36 = 1102.61; p≤ 0.000001), NaCl (F_2,36 = 859.49; p≤ 0.000001), humates (F_1,36= 844.90; p≤ 0.000001 ) and the interaction of NaCl × humates (F_2,36 = 4.69; p≤ 0.001). For interactions varieties × NaCl, varieties × humates and varieties × NaCl × humates, showed no significant differences. When analyzing interactions, it was observed that in all three napoletano variety in the three NaCl concentrations, showed the highest stem length, which decreased in both varieties as salinity levels increased (Table 1). In both varieties, stem length increased when humate was applied, being slightly higher in napoletano both control treatment and dilution 1/60 (Table 2). Although the interaction varieties × NaCl × humates showed no significant differences, Napoletano showed the highest LT in 0 mM NaCl and humate dilution of 1/60, also in both varieties, LT increased compared to control in humate dilution 1/60 at all NaCl concentrations. The lowest LT was for the variety Sweet Genovese in 100 mM NaCl and 0 humate (Table 3).

Humate concentration increased plant height (Table 2) and these dilutions counteracted saline stress, ever since dilution 1/60 (v/v) mitigated the negative effect of salinity. This coincides with ^{Fernandez-Luqueño et al., (2010)} who found that bean plants treated with vermicompost, showed higher growth to one third compared to controls in plant height. A similar responses found ^{Channabasanagowda et al. (2008)} on wheat plants with application of vermicompost, noting a significant increase in height. ^{Buniselli et al. (1990)} working with corn, found an increase in weight, plant height, ear length and grain yield when applied humic acids. Shovelful et al. (1999) evaluated basil plants both in ground and in pots with various substrates, finding that it develops better under port conditions than in ground conditions and the best substrate was the organic substrate (mixture of manure and wheat straw); similar results were detected by ^{Yao Lei et al. (2000) Nardi et al. (2002b)}; and ^{Miceli et al. (2003)}.

Radicle length (LR) showed significant differences between varieties (F_1,36 = 426.0; p≤ 0.0001), NaCl (F_2,36 = 388.16; p≤ 0.0001), humates (F_1,36 = 379.35; p≤ 0.0001) interactions varieties × NaCl (F_2,36 = 9.02; p ≤ 0.0001), and varieties × humates (F_1,36 = 33.80; p≤ 0.0001), NaCl × humates (F_2,36 = 11.05; p≤ 0.0001) and varieties × NaCl × humates (F = _2.36 = 12.95; p≤ 0.001). The analysis of the main interactions revealed that Napoletano variety showed the highest LR in all NaCl concentrations, decreasing linearly in both varieties as NaCl levels increased (Table 1). Napoletano showed greater LR both control and humate dilution of 1/60 and in both varieties increased with the application of humates (Table 2).

The interaction varieties × NaCl × humates showed Napoletano with the highest LR in 0 mM NaCl and humate dilution 1/60; also, in both varieties, LR increased over control in humate dilution 1/60 dilution at all NaCl concentrations. The lowest LR was for Sweet Genovese in 100 mM NaCl and 0 humate (Table 3). The results indicate an increase in LR from the application of humates. While root development is severely affected by salt stress, humic acids from humates of vermicompost positively influenced growth. This is because humic acids promote increases in permeability of cell membrane in basil, so despite hypertensive saline conditions, favored seed imbibition, to solubilize starch, thus favoring carbohydrate availability for root growth. Other effect is due to a function as a regulator or growth promoter, inducing growth, as noted by ^{Nardi et al., (2002a)}.

The results of this study are consistent with those of ^{Barros et al., (2010)} who noted that humic substances as abscisic acid and indole-acetic acid contained in vermicompost, promote plant growth. Meanwhile, ^{Ermakov et al. (2000)} found that the humic acid adsorbed to cells of the plant at surface level, increases its permeability, which promotes nutrient absorption by plants. The initial negative effect observed in seedling growth in saline environments, is counteracted by the action of humic substances applied, inducing a sudden growth later (^{Yldrm et al., (2003)}.

Fresh root biomass (BFR) showed significant differences between varieties (F_1,36 = 373.65; p≤ 0.0001), NaCl (F_2,36 = 682.28; p≤ 0.0001), humates (F_1,36 = 689.89; p≤ 0.0001), the interaction varieties × humates (F_1,36 = 42.07; p≤ 0.0001) and varieties × NaCl × humates (F = _2.36 = 7.93; p≤ 0.001). For interactions varieties × NaCl and NaCl × humates showed no significant differences. The analysis of significant interactions of factors, showed napoletano with the highest BFR at all NaCl concentrations, which decreased in both varieties as salinity levels increased (Table 1). The Neapoletano variety also showed greater BFR both in control and humate dilution 1/60, noting that both varieties increased BFR in humate dilution 1/60 (Table 2).

Although the interaction varieties × NaCl × humates showed no significant differences, Napoletano showed the highest BFR in 0 mM NaCl and humate dilution 1/60; moreover, in both varieties, BFR increased over the control in humate dilution 1/60 at all NaCl concentrations. The lowest BFR was for Sweet Genovese in 100 mM NaCl and 0 humate (Table 3). Meanwhile root dry biomass (BSR) showed significant differences between varieties (F_1,36 = 408.64; p≤ 0.0001), NaCl (F_2,36 = 449.59; p≤ 0.0001), humates (F_1,36 = 373.83; p≤ 0.0001) and interaction NaCl × humates (F_1,36 = 3.75; p≤ 0.0001). For interactions varieties × NaCl, varieties × humates and triple interaction varieties x NaCl × humates, showed no significant differences. Table 1 shows that Napoletano variety had higher BSR values, which decreased in both varieties as salinity levels increased. Also Napoletano showed the highest BSR both in control and humate dilution 1/60 and in both varieties BSR increased with humate application (Table 2).

Although the interaction varieties × NaCl ×humates showed no significant differences, Napoletano showed greater BSR at 0 mM NaCl and humate dilution 1/60; also, in both varieties BSR increased over the control in humate dilution 1/60 at all NaCl concentrations. The lowest BSR was for Sweet Genovese in 100 mM NaCl and 0 humate (Table 3). Fresh and dry root biomass is manifested by an increase of biomass with the application of humates vermicompost and exhibits a linear decrease in function of increasing levels of NaCl. These results coincide with those from ^{Fernández-Luqueño et al. (2010)} who conducted an experiment on bean plants to determine if humic substances increase biomass, finding a double increase of fresh and dry root biomass, compared to control treatment.

Moreover, ^{Chen and Aviad (1990)} consider that humic substances, improve the root formation and growth, thus dry biomass. Fresh stem biomass (BFT) showed significant differences between varieties (F_1,36 = 1341.42; p≤ 0.0001), NaCl (F_2,36 = 687.63; p≤ 0.0001), humates (F_1,36 = 424.0; p≤ 0.0001) , interactions varieties × NaCl (F_2,36 = 5.66; p≤ 0.0001), varieties × humates (F_1,36 = 5.30; p≤ 0.0001), NaCl × humates (F_2,36 = 3.63; p≤ 0.0001). For the triple interaction varieties × NaCl × humates showed no significant differences. Table 1 shows that Napoletano variety had the highest BFT in the three NaCl concentrations. BFT decreased in both varieties as salinity levels increased (Table 1).

Napoletano also showed greater BFT both in control and humate dilution 1/60, noting that this in both varieties this variable increased by adding humates (Table 2). Although the interaction varieties × NaCl × humates showed no significant differences, Napoletano showed greater BFT in 0 mM NaCl and humate dilution 1/60; moreover, in both varieties, BFT increased over the control in humate dilution 1/60 all NaCl concentrations. The lowest BFT was for Sweet Genovese in 100 mM NaCl and 0 humate (Table 3).

Stem dry biomass (BST) showed significant differences between varieties (F_1,36 = 3214.99; p≤ 0.0001), NaCl (F_2,36 = 1181.10; p≤ 0.0001), humates (F_1,36 = 1000.26; p≤ 0.0001), interactions varieties × NaCl (F_2,36 = 29.70; p≤ 0.0001), varieties × humates (F_1,36 = 9.00; p≤ 0.0001), NaCl × humates (F_2,36 = 16.71; p≤ 0.0001) and varieties × NaCl × humates (F=_2,36 = 11.19; p≤ 0.0001). Table 1 shows that Napoletano variety exhibited the highest BST, which decreased in both varieties as salinity levels increased. Napoletano also showed higher BST both in control and humate of vermicompost dilution 1/60, observing that for both varieties, BST increased when applying humate (Table 2).

The interaction variety × NaCl × humates showed Napoletano with higher BST in 0 mM NaCl and humate dilution 1/60; also, in both varieties, BST increased regarding control at humate dilution 1/60 at all NaCl concentrations. The lowest BST was for Sweet Genovese in 100 mM NaCl and 0 humate (Table 3). Fresh leaf biomass (BFH) showed significant differences between varieties (F_1,36 = 1672.17; p≤ 0.0001), NaCl (F_2,36 = 913.80; p≤ 0.0001), humates (F_1,36 = 492.24; p≤ 0.0001 ), interactions varieties × NaCl (F_2,36 = 19.32; p≤ 0.0001) and NaCl × humates (F_2,36 = 9.40; p≤ 0.0001). For interaction varieties × humates and varieties × NaCl × humate showed no significant differences.

Table 1 shows that Napoletano had the highest BFH, which decreased in both varieties as salinity levels increased. Napoletano also showed higher BFH both in control and humate dilution 1/60, noting that in both varieties, BFH increased by applying humates (Table 2). Napoletano showed higher BFH in 0 mM NaCl and humate dilution 1/60; moreover, in both varieties, BFH increased with respect to control at humate dilution 1/60 at all NaCle concentrations. The lowest BFH was for Sweet Genovese 100 mM NaCl and 0 humate (Table 3).

Leaf dry biomass (BSH) showed significant differences (F_1,36 = 1765.63; p≤ 0.0001), NaCl (F_2,36 = 883.24, p≤ 0.0001), humates (F_1,36 = 633.13; p≤ 0.0001), interactions varieties × NaCl (F_2,36 = 16.93; p≤ 0.0001), NaCl × humates (F_2,36 = 11.60; p≤ 0.0001) and varieties × NaCl × humates (F = _2.36 = 14.58; p≤ 0.0001). Interaction varieties × humates showed no significant differences. Table 1 shows that Napoletano showed higher BSH, which decreased in both varieties as salinity levels increased. Napoletano showed higher BSH in both the control and humate vermicompost dilution of 1/60; however, it was observed that both varieties BSH increased when humate was applied (Table 2).

Interaction varieties × NaCl × humates showed Napoletano with the highest BSH in 0 mM NaCl and humate dilution 1/60 and in both varieties, BSH increased with respect to control in humate dilution 1/60 at all NaCl concentrations. The lowest BSH was for Sweet Genovese 100 mM NaCl and 0 humate (Table 3). The response of fresh and dry biomass of stem and leaf to humates vermicompost showed higher values with the application of humate, suggesting that this tends to mitigate the negative effect of saline stress, which manifest in biomass production. ^{Degano (1999)} tested two sources of salt (NaCl and NaSO₄) on growth and fresh stem weight of Tessaria absinthoides, finding that as salt concentration increased, regardless of source, fresh biomass decreased, ascribing this decrease to the distance and length of internodes had this same response, giving the plant a short appearance.

This response is attributed to the osmotic effect which originates in salt solution, same that difficult the water system of the plants and the toxic effect of interfering ions in metabolic processes such as carbohydrate synthesis and transport of photosynthetic products, thus its use in the production of new tissue, in addition to a positive effect on microbiological components (^{Gutiérrez et al., 2008}), plant hormones (^{Jana et al., 2010}), available ions (^{Sallaku et al., 2009}) and modification of the physico-chemical properties of soil (^{Azarmi et al., 2008}).

Conclusions

There is a differential response between varieties for morphometric variables stem length and root, fresh and dry biomass of root, stem, leaf and leaf area in salt stress conditions and the application of humate vermicompost, highlighting the Napoletano variety as the most tolerant, exhibiting the highest values in all variables with the application of bio-stimulant. The use of humates of vermicompost stimulated morphometric variables stem length and root, fresh and dry biomass of root, stem, leaf and leaf area of basil varieties under saline conditions, allowing that the tolerant variety to improve its emergence and growth and the sensitive variety increase its tolerance to salt stress.