Arbuscular mycorrhizal symbiosis in the maximization of the concentration of foliar biomolecules in pomegranate ( Punica granatum L . ) seedlings

The pomegranate (Punica granatum) is a fruit tree with medicinal potential due to the presence of bioactive compounds with pharmacological characteristics. Arbuscular mycorrhizal fungi (AMF), when in symbiosis, optimize the production of metabolites with medicinal potential. The objective of this study was to select efficient AMF to maximize the growth and the production of foliar metabolites with medicinal characteristics in P. granatum seedlings. The experimental design was completely randomized, with three inoculation treatments (inoculated with Gigaspora albida, inoculated with Acaulospora longula and a non-inoculated control) and 7 repetitions, totaling 21 experimental units. Pomegranate plantlets with two definitive leaves were transferred to pots with soil containing 3 mg P dm and were inoculated at the root region with soil-inoculum supplying 200 spores/pot of G. albida or A. longula. 120 days after inoculation, the following parameters were evaluated: height, number of leaves, stem diameter, fresh and dry matter of the aerial part and fresh matter of the subterranean part and mycorrhizal colonization. On the leaves, the concentration of total proteins, soluble carbohydrates, total phenols, total tannins and total flavonoids was determined. Pomegranate seedlings associated with G. albida accumulate more proteins (213, 36%), carbohydrates (246, 75%), phenols (198, 40%), flavonoids (410, 81%) and tannins (59, 92%) compared to the non-inoculated control. Similar behavior was found for the other parameters of vegetal growth, with the exception of stem growth and of the mycorrhizal colonization. It can be concluded that pomegranate seedlings benefit from inoculation with AMF in terms of growth and production of bioactive compounds, depending on the species of fungus that was used.


INTRODUCTION
Plant species can produce secondary metabolic compounds which are used as alternative resources for treatments (Fenner et al., 2006); some species can serve as raw material at low cost for the production of medicine, and biotechnological actions are needed to maximize the phytomass production for the pharmaceutical industry.The pomegranate (Punica granatum) contains various active metabolites that can act in the treatment of diseases (Pereira et al., 2006).The metabolites found in the phytomass of this plant show hypoglycemic, antibacterial (Lansky and Newman, 2007) and anti-inflammatory and analgesic activities (Werkman et al., 2008), apart from the potential of treating gastro-intestinal (Matos, 1989) and neoplasic (Khan, 2009;Pereira et al., 2005) diseases.
Among the microorganisms that are present in the soil, arbuscular mycorrhizal fungi (AMF) form a mutualistic symbiotic association with the roots of plant species (Smith and Read, 2008) that stimulate the growth and the productivity of economically interesting crops (Oliveira et al., 2013).Besides the nutritional benefits, mycorrhization can increase the production of bioactive compounds of plants with pharmacological characteristics.Studies have shown that the various molecules of the primary and secondary metabolism optimize their production when the plants are associated with AMF (Pedone-Bonfim et al., 2013;Huang et al., 2011;Ceccarelli et al., 2010;Ratti et al., 2010;Rapparini et al., 1996).
In micropagated seedlings of P. granatum inoculated with AMF, Singh et al. (2012) reported an increase in the production of phenolic compounds compared to the uninoculated control.However, considering the phytotherapeutic importance, it has not been established whether the inoculation with AMF promotes the accumulation of other foliar biomolecules in P. granatum seedlings, as flavonoids, tannins and proteins.
Therefore, the hypothesis tested that mycorrhization favors the production of primary and secondary metabolites in the leaves of pomegranate seedlings and the mycorrhizal efficiency depends on the fungus that was used.
The objective of this study was to select efficient AMF to maximize the growth and the production of foliar metabolites with medicinal characteristics in P. granatum seedlings.

Arbuscular mycorrhizal fungi (AMF)
Acaulospora longula Spain & N.C.Schenck (UFPE 21) and Gigaspora albida N. Schenck & G.S. Sm. (UFPE 01) were tested.The AMF inocula used were produced in a soil which had millet (Panicum miliaceum L.) as a host and were stocked at 4°C until the moment of their use.

Mycorrhizal inoculation
Plantlets with two definitive leaves were transferred to the pots and inoculated at the root region with soil-inoculum supplying 200 spores + hyphae + colonized roots of A. longula or G. albida.The experiment was kept in a greenhouse at Universidade do Estado da Bahia, Brazil.After 120 days of inoculation the following parameters were evaluated: height, stem diameter, number of leaves, fresh matter of the aerial part, fresh matter of the subterranean part, dry matter of the aerial part and mycorrhizal colonization.To evaluate the mycorrhizal colonization, the roots were clarified (KOH 10%) and stained (Trypan blue 0.05%, v/v) by the Phillips and Hayman (1970) method, and the colonization percentage was determined by the gridline intersection method (Giovannetti and Mosse, 1980).

Biochemical determinations
The concentration of total proteins, soluble carbohydrates, total phenols, total tannins and total flavonoids was determined in the leaves.The preparation of the foliar extract was carried out after drying (45°C) and 500 mg of leaves were punctured and transferred to Erlenmeyers (125 ml) and 20 ml of ethanol (95%) was added.After 12 days of maceration (25°C), in the dark, the extract was filtered in gauze and refiltered in a qualitative filter paper, and stocked in ambar flasks (-4°C) (Brito et al., 2008).The following molecules in the extract were determined: total proteins by the Bradford (1976) method, soluble carbohydrates estimated by the Dubois et al. (1956) method, total phenols in accordance with the methodology proposed by Monteiro et al. (2006), total tannins by the casein precipitation method, described by Monteiro et al. (2006) and total flavonoids estimated based on the methodology of Araújo et al. (2008).The experimental design was completely randomized with three inoculation treatments (inoculated with G. albida, inoculated with A. longula and a non-inoculated control), with seven repetitions, totalizing 21 experimental units.The data were submitted to analysis of variance (ANOVA) and the averages compared to the Table 1.Concentration of total proteins, soluble carbohydrates, total phenols, total flavonoids, total tannins in pomegranate seedlings, associated or not with arbuscular mycorrhizal fungi, 120 days after inoculation, in greenhouse.); **(µg g plant -1

Variable
). Averages (n= 7) followed by the same letter do not differ from the Tukey test (1 %).

RESULTS AND DISCUSSION
With the exception of the stem diameter, the inoculation treatments had an effect on the studied variables (p < 0.01).Pomegranate seedlings associated with G. albida produced more metabolites compared to the plants inoculated with A. longula (Table 1).P. granatum seedlings associated with G. albida accumulate proteins (213, 36%), carbohydrates (246, 75%), phenols (198, 40%), flavonoids (410, 81%) and tannins (59, 92%) compared to the non-inoculated control.In other situations, the application of mycorrhizal technology favored the maximization of the production of bioactive compounds with medicinal potential (Huang et al., 2011;Ratti et al., 2010).It is likely that the increase of metabolite levels is associated with the biochemical changes in the plastids and mitochondria (Strack and Fester, 2006), the efficiency in the metabolic routes involved in the biosynthesis of the compounds that were studied as suggested by Mandal et al. (2013), as well as an improvement in the nutritional state (Oliveira et al., 2013).Another factor that has to be studied is the variance in production of the studied compounds in relation to the mycorrhizal treatment, as was shown by Dave and Tarafdar (2001) in Ocimum basilicum seedlings and by Singh et al. (2012) in P. granatum seedlings, thereby confirming the initial working hypothesis and indicating that generalizations about the mycorrhizal efficiency in the production of plant metabolites should be avoided.Araim et al. (2009), when evaluating the effect of mycorrhization with Glomus intraradices in Echinacea purpurea plants found that there was an increase in the protein concentrations, and pointed out that the symbiosis plays an important role in the maximization of the production routes of metabolites, as was confirmed in the present study (Table 1).The secondary metabolism, translated by the production of phenolic compounds, was favored by the mycorrhization of pomegranate seedlings with G. albida (Table 1).With similar results, Kapoor et al. (2007) also related that the mycorrhization of Artemisia annua by Glomus macracarpum and Glomus fasciculatum favored the production of artemisinin.Ponce et al. (2004) also documented similar behavior in Trifolium repens inoculated with G. intraradices.On the other hand, mycorrhization with Glomus mosseae did not alter the accumulation of essential oils in Origanum sp.seedlings (Khaosaad et al., 2006), which was a similar result to what was verified in pomegranate seedlings forming symbiosis with A. longula (Table 1).
Seedling leaves of P. garantum inoculated with G. albida accumulated 93.40 µg phenols/g plant, a concentration higher than reported by Singh et al. (2012) in micropropagated plants of this species, therefore, the biotechnology system employing G. albida is alternative to increase the production of phenolics and favors the synthesis of other plant molecules (Table 1).
The mycorrhizal inoculation favored the growth of inoculated pomegranate seedlings with G. albida for the majority of the evaluated parameters (Table 2).Similar results were described by Cavender et al. (2003), who found satisfactory results in the dry matter of the aerial and subterranean part in Sorghum bicolor inoculated with mycorrhizal fungi.Bai et al. (2008) showed an increase in the dry matter of the aerial part in Zea mays (corn) inoculated with Glomus caledonium, Glomus ssp.and Acaulospora ssp.Besides this, benefits of the utilization of AMF in the optimization of plant growth were registered in other studies (Sheng et al., 2008;Machineski et al., 2009;Sharma et al., 2009;Pedone-Bonfim et al., 2013) In a similar way, as concerns these biochemical and phytochemical findings, mycorrhization of seedlings with G. albida produced differentiated benefits in relation to the non-inoculated plants or those associated with the other tested AMF (Table 1).It is likely that greater functional compatibility has occurred between the Gigasporaceae and the plant that was studied.Such a fact has been documented (Souza et al., 2010).Besides this, it is known that members of Gigaspora produce profuse extraradicular mycelium in the soil (Hart and Reader, 2002), which may have contributed to the greater benefits registered in this treatment.However, to prove such a hypothesis, it is necessary to quantify the extraradicular mycelium, which has to be realized through tests using sterilized soil, and the current experiment was conducted in non-disinfected.
In the non-inoculated control treatment, there was a formation of mycorhizal structures at the root cortex of pomegranate seedlings (Table 2) and the highest levels of mycorrhizal colonization were found in plants associated with A. longula (Table 2).In this case, the occupation of the cortex by AMF was not translated into growth increments and the bioaccumulation of the studied molecules (Tables 1 and 2).Divergent results were registered by Silva (2006), who found that Passiflora alata seedlings grew more when the root cortex was more occupied by mycorrhizal structures, especially arbuscules.Studies must be conducted to verify the benefit of the inoculation registered in this study, under field conditions, in this way validating the mycorrhizal-technology for the production optimization of phytochemicals in pomegranate.

Conclusion
The growth and the production of biomolecules in pomegranate seedlings are optimized by inoculation with AMF with the benefits varying according to the fungus that was used.The symbiosis between pomegranate seedlings and G. albida is a biotechnological alternative for the production of seedlings with increased levels of primary and secondary foliar metabolites with medicinal properties.

Table 2 .
Number of leaves, stem diameter, fresh matter of the aerial part, dry matter of the aerial part, fresh matter of the subterranean part, height and mycorrhizal colonization in pomegranate seedlings, associated or not with arbuscular mycorrhizal fungi, 120 days after inoculation, in greenhouse.
a Averages (n= 7) followed by the same letter do not differ from the Tukey test (1%).