Of the 25,000 species of orchids in the world, Mexico is home to around 1,260 species belonging to 170 genera (Hágsater et al., 2005); however, it is necessary to promote knowledge of their management to encourage the introduction of these native plants in Mexican floriculture.
Orchids account for around 10 % of the international flower trade, with a commercial value, from 2007 to 2012, of US$ 483 million; likewise, in 2012 there were more than 40 countries exporting orchids and 60 importing them, with the overall value of the global trade totaling US$ 504 million (Lakshman, Pathak, Rao, & Rajeevan, 2014). This volume of production involves costs of various kinds, among which are the fertilizers and the costs generated by their application. Therefore, ornamental horticulture, like any agricultural activity, requires the rational use of chemical fertilizers and the use of alternative sources to make it more environmentally friendly (Daughtrey & Benson, 2005), as in the case of biofertilizers (Boraste et al., 2009).
While there are studies on the use of mineral fertilizers in orchid nutrition (Bichsel & Starman, 2008; Wang, 2000; Wang, 1996; Zong-min, Ning, Shu-yun, & Hong, 2012), there are few specific ones and the application form of many others is unknown. Among the advantages of the efficient use of fertilizers are the stimulation of vegetative growth, the increase of precocity in flowering and the promotion of the symbiotic relationship with mycorrhizal fungi (Espinosa-Moreno, Gaytán-Acuña, Becerril-Román, Contreras, & Trejo-López, 2000).
On the other hand, the effectiveness of using biofertilizers in ornamental plants has been reported (Abbasniayzare, Sedaghathoor, & Dahkaei, 2012; El-Sayed, Shahin, & Zaky, 2012; Scagel, 2003); however, information about their use in orchids is scarce. A biofertilizer is defined as a substance that contains living organisms, which when applied to the surface of the plant or to the soil colonize the rhizosphere or the interior of the tissues of the plant and promote its growth by increasing the supply or availability of nutrients (Amanullah, Kurd, Khan, Ahmed, & Khan, 2012; Youssef & Eissa, 2014). The most noteworthy microorganisms present in a biofertilizer are arbuscular mycorrhizal fungi (AMF) and plant growth promoting rhizobacteria (PGPR) (Boraste et al., 2009).
The factors that determine the physiological quality of plants are those related to photosynthesis (fluorescence of photosynthetic pigments, stomatal conductance, rate of photosynthesis, concentration of chlorophylls, etc.), concentration of nutrients and reserve sugars, among others (Villar, 2003). Also, some authors report that one of the most important nutrients is nitrogen, since it is involved in the formation of amino acids, proteins and other cellular constituents (Trejo-Téllez, Gómez, Rodríguez, & Alcántar, 2005). Ling and Subramaniam (2007) analyzed the content of chlorophylls and proteins in Phalaenopsis violacea (Orchidaceae) to determine quality aspects in tissue culture, for breeding purposes. Hew and Yong (1994) reported an analysis of growth and photosynthesis, where they included chlorophyll content in Oncidium 'Goldiana', a hybrid used for the cut flower industry.
Therefore, the objective of this work was to evaluate the effect of mineral fertilization and biofertilization on some physiological parameters of Laelia anceps Lindl. subsp. anceps seedlings in vegetative stage.
Materials and methods
This research was carried out under greenhouse conditions at the Colegio de Posgraduados, Montecillo Campus. Two-year-old Laelia anceps Lindl. subsp. anceps seedlings propagated in vitro by seed and averaging 8 cm in height were used. The transplant was made from trays to 340-mL opaque plastic containers, which contained a mixture of pine bark and perlite at a ratio of 75:25 (%, v/v) with a particle size of 4-6 and 2-3 mm, respectively.
Nutrient solutions. Different nutrient solutions, formulated from different sources of nutrients (Table 1), were applied during irrigation.
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Treatments. 1) Nutrient solution formulated from mineral fertilization (MF), 2) biofertilization (BFERT), 3) biofertilization as the main source of the solution supplemented with minerals (MF + BFERT) and 4) control plants irrigated with distilled water (C). Treatments 1 to 3 were supplemented with micronutrients.
The nutrients and their concentration (mg·L-1), in the solutions of treatments 1 to 3, were: N (225), P (75), K (75), Ca (25), Mg (12.5), Fe (1.9), Cu (0.375), Zn (0.75), Mn (0.37) and B (0.15). Additionally, biofertilization (treatments 2 and 3) contained: humic acids (15 %), amino acids (0.64 %), indole-3-butyric acid (98 mg·L-1), Bacillus subtilis (200 x 10⁵ CFU·L-1), Azospirillum brasilensis (40 x 10⁵ CFU·L-1) and Glomus intraradices (6,000 spores·g-1).
The pH of the solutions was adjusted to 5.5 with 1 N H2SO2, for which a portable potentiometer (PC18, Conductronic, Mexico) was used. Application of the nutrient solutions (30 mL per container) was carried out weekly from the third week after transplant. The spatiality of the application was based on the size of the seedlings under study, size of the container, amount of N in the nutrient solutions, specific observations of the species (wild material, not adapted to ornamental culture and roots sensitive to salinity) and recommended orchid fertilization intervals (Chang, Wu, & Hsieh, 2010; Lima-do Amaral et al., 2010; Rodrigues, Ferreira-Novais, Alvarez, Moreira-Dias, & de Albuquerque-Villan, 2010; American Orchid Society, 2017). After each fertilization, three irrigations were made weekly with 200 mL of water each to avoid accumulation of salts and desiccation in the substrate.
Study variables. In both leaves and pseudobulbs, the concentration and content of chlorophylls were assessed, along with the concentration of amino acids and total soluble proteins.
Concentration of chlorophylls a, b and total (mg·g-1 fresh weight [FW]). It was determined using the method described by Harborne (1973). The samples were read in a spectrophotometer (Genesys 10 UV, Thermo Fisher Scientific, USA) with an absorbance of 663 and 645 nm.
Content of chlorophylls a, b and total (mg·plant-1). It was estimated from the concentration values obtained from each biomolecule in leaves and pseudobulbs and by considering the fresh matter weights of these organs per plant.
Concentration of total soluble amino acids (μM·g-1 FW). They were determined by ethanolic extraction (Geiger et al., 1998) with the ninhydrin method (Moore & Stein, 1954). Leucine was used to make the standard curve at a concentration of 0 to 250 mM. The extracts were read in a spectrophotometer (Genesys 10 UV, Thermo Fisher Scientific, USA) at 570 nm.
Concentration of total soluble proteins (μg·g-1 FW). Protein extraction was performed on leaves and pseudobulbs at the time of cutting, according to the method described by Höfner, Vázquez-Moreno, Abou-Mandour, Bohnert, and Schmitt (1989).
The experiment was established under a completely randomized design with 15 replicates. The experimental unit consisted of a container with a plant. With the obtained data, an analysis of variance was made with the Statistical Analysis System statistical package (SAS Institute, 2010). Tukey’s range test (P ≤ 0.05) was also performed.
Results and discussion
Concentration and content of chlorophylls
The concentration of chlorophyll a in pseudobulbs was significantly higher (P = 0.007) in the MF and BFERT treatments, compared to MF + BFERT. A similar trend was observed in chlorophyll b (P = 0.048) and total chlorophylls (P = 0.020) (Figure 1A). In leaves, the applied fertilization treatments showed no significant statistical differences among treatments in the different types of chlorophyll: a (P = 0.360), b (P = 0.125) and total (P = 0.161) (Figure 1A).
In leaves and pseudobulbs, the chlorophyll a and b contents were significantly higher (P ≤ 0.05) with MF and BFERT, compared to MF + BFERT and C plants. As for total chlorophylls in leaves, only the BFERT significantly increased its content (P = 0.0009), while in pseudobulbs, both MF and BFERT gave statistically higher values (P = 0.0002). In both organs, the chlorophyll b and total contents with MF + BFERT were significantly lower than the C plants (Figure 1B). Ling and Subramaniam (2007) found concentrations of total chlorophylls in Phalaenopsis violacea up to 0.448 mg·g-1, which were higher than the highest found with the MF and BFERT treatments for L. anceps subsp. anceps (from 0.091 to 0.176 mg·g-1). Likewise, Trelka, Włodzimierz, Jóźwiak, and Kozłowska (2010) reported higher concentrations of chlorophyll a (from 0.25 to 0.3 mg·g-1) and b (around 0.1 mg·g-1) in leaves of the Phalaneopsis orchid 'Sprigfield'. In Laelia anceps subsp. anceps, the concentration of chlorophyll a in leaves was higher than that reported by Hew and Yong (1994) for Oncidium ‘Goldiana’ (0.071 mg·g-1).
The results showed that the chlorophyll a, b and total contents in L. anceps subsp. anceps pseudobulbs, although they were smaller than in leaves, increased with MF and BFERT. Leaves are the main photosynthetic organs of plants; in addition, in orchids other non-foliar organs possess chlorophyll and are capable of fixing carbon dioxide, such as pseudobulbs, roots, flowers and capsules (Hew & Yong, 2004; Ng & Hew, 2000). The presence of chlorophylls in the various organs of the plant indicates that they also carry out photosynthesis (Vermaas, 1998). Additionally, the content of chlorophylls is closely related to the total nitrogen present in plant tissues, and, therefore, is an indirect indicator of the nutrient status of the plant (Zarco-Tejada et al., 2004).
Concentration of amino acids
In pseudobulbs, the MF significantly increased (P = 0.0001) the concentration of amino acids in relation to the rest of the treatments (Figure 2), while in leaves, fertilization treatments did not significantly (P = 0.549) affect the concentration of amino acids.
Concentration of proteins
In leaves, the concentration of proteins was significantly higher (P = 0.058) with the MF + BFERT treatment than with the control. In pseudobulbs, there were no statistical differences due to the effect of treatments (Figure 3). The foliar concentration of proteins for Laelia anceps subsp. anceps was lower than that reported by Ling and Subramaniam (2007) for Phalaenopsis violacea (55 µg·g-1 FW). In several studies, the increase in total proteins has been observed in response to inoculation treatments of microorganisms or products derived from them, which in turn is related to the crop growth and development (González-Vega, Hernández-Rodríguez, Barrios-Alonso, Velázquez-del Valle, & Hernández-Lauzardo, 2007).
In the present work, the protein concentration in leaves with BFERT showed a similar effect to that of MF. These results contrast with what was found by González, Cabrera, and Hernández (2002), who used RIZOBAC® microorganisms as bio-fertilizer in coffee plantlets and found that they had a higher concentration of total proteins than the control.
The mineral fertilization and biofertilization applied individually increased the concentration of chlorophylls a, b and total in pseudobulbs, and their content in leaves and pseudobulbs. Mineral fertilization significantly increased the concentration of amino acids in pseudobulbs, and its combination with biofertilization increased the foliar concentration of proteins. The increase in the concentration of biomolecules such as chlorophylls, amino acids and proteins are directly related to plant growth, which in turn is an indicator of basic plant processes, such as photosynthesis, respiration and transpiration. It can thus be concluded that biofertilizers have potential for use in Laelia anceps subsp. anceps.