Abstract
Passiflora setacea DC. growing is of interest to the herbal industries since in its leaves are produced secondary metabolites that confer antioxidant, anxiolytic, and antidepressant properties in Passiflora. Therefore, it is important to search for sustainable alternatives that aim to enhance the production of these compounds to add value to the phytomass, such as the inoculation with arbuscular mycorrhizal fungi (AMF) and the application of coconut coir dust, which has not been reported to P. setacea yet. The aim was to select the efficient combination of AMF and coconut coir dust to increase the compounds’ production and optimize the antioxidant activity in P. setacea leaves. The P. setacea seedlings that were cultivated in substrates without coconut coir dust and colonized by Gigaspora albida N.C. Schenck & G.S. Sm. produced more total saponins (1,707.43%), total tannins (469.98%), and total phenols (85.81%), in comparison to the non-mycorrhizal plants, in addition to enhancing the glomalin-related soil proteins. On the other hand, in general, the use of coir dust as a substrate has not been shown to increase the production of these bioactive compounds. It is concluded that the production of P. setacea seedlings using G. albida is an alternative to offer phytomass to the herbal medicines industry based on passion fruit.
Keywords: Mycorrhization, Organic substrates, Passion fruit, Phenolic compounds, Saponins
Introduction
The Passiflora phytomass is used in herbal medicines [1, 2], since the leaves have anxiolytic [3, 4], sedative [5], and antidepressant properties [6]. These benefits are attributed to the biomolecules, highlighting the isorientin and vitexin, which are the main phenolics found in Passifloraceae [7] and known to grant calming effects [8]. Furthermore, the saponins present in the passion fruit are capable to enhance the anxiolytic effects [9].
Every year about 600.000 tons of passion fruits in Brazil is produced [10], and this country is considered the largest worldwide producer of this fruit tree [11]. Although there are more than 150 passion fruit occurring species in Brazil [12], only three are cultivated and have a commercial expansion, the yellow passion fruit (Passiflora edulis f. flavicarpa Deg), the sweet passion fruit (Passiflora alata Curtis), and the purple passion fruit (Passiflora edulis f. edulis Sims), in which part of this cultivation is destined to the production of anxiolytic medicines.
Another passion fruit species that has agronomic potential is the Passiflora setacea DC. This species, aside from concentrated vitexin [7], has resistance to soil pathogen microorganisms [13], and phytopathogens [14]; with such characteristics, P. setacea has the potential to be incorporated into the market of raw materials to produce anxiolytic herbal medicines.
However, to be attractive to the herbal medicine industry, it is necessary to develop sustainable technologies that increase the plant productivity and the concentration of foliar biomolecules of interest in the phytomass, to encourage the producers. In this regard, the application of arbuscular mycorrhizal fungi (AMF) and organic substrates is promising, considering that the mycorrhizal technology can increase biomolecules production in fruit trees [15, 16]. In this way, the coconut coir dust is obtained by the coconut mesocarp; it is known for improving the soil’s physical properties and the electric conductivity and it does not contain phytopathogens [17]; when it is applied properly, it increases the biomolecules production in P. edulis [18].
However, these benefits have not been studied in P. setacea yet. The only study about the AMF effect in P. setacea reported the increase of macro and micronutrients absorption [19]. In other passion fruit species, such as P. alata and the P. edulis, the mycorrhization associated with organic fertilizers promoted the increment in the foliar biomolecules concentration and the growth parameters [18, 20–22].
Therefore, it was tested the hypothesis that the AMF inoculation associated with the coconut coir dust supply optimizes the phenolic compound production and the antioxidant activity in P. setacea seedlings. Thus, this study aimed to select the efficient combination of AMF and coconut coir dust to increase the production of the compounds and optimize the antioxidant activity in leaves of P. setacea seedlings.
Material and methods
The experiment was conducted in a greenhouse (Biological Sciences Institute, University of Pernambuco, Recife, PE — Brazil). After 68 days, the plants were harvested, since the tendrils had emerged, the growth parameters, the mycorrhizal activity, and the biomolecules production were evaluated at the Analysis Laboratory, Research and Studies on Mycorrhizae — LAPEM/UPE.
AMF inoculum and substrates
The Laboratory of Mycorrhizae of the Department of Mycology of the Federal University of Pernambuco, Brazil, granted the initial soil inoculum utilized in this research. The isolates of Acaulospora longula Spain & N.C. Schenck and Gigaspora albida N.C. Schenck & G.S. Sm. were multiplied in soil, using Panicum milliaceum L. as the host plant. After 90 days, the inocula were collected and stored at 4 °C until using and presented 60% of the mean infection percentage [23].
The sand (Areiasil®, Sirinhaem, Pernambuco, Brazil), which was autoclaved twice (121 °C; 15 min) and washed, the seedling substrate (Viva o Verde®, Recife, Pernambuco, Brazil), and the coconut coir dust (Viva o Verde®, Recife, Pernambuco, Brazil) were mixed at the chosen proportions and transferred to seedling polybags in three types of coir-based substrates (S): S1 (sand + substrate + coconut coir dust (1:1:1), S2 (sand + substrate + coconut coir dust (3.5:3.5:2), and S3 (sand + substrate (1:1); thus, S3 was the control condition, since no coir dust was added (Table 1).
Table 1.
Chemical characterization of the substrates used in the experiment with Passiflora setacea DC.
| Substrates | pH | P | Ca | Mg | Al | K |
|---|---|---|---|---|---|---|
| (H2O,1:2.5) | mg dm−3 | cmolc dm−3 | ||||
| S1a | 5.10 | 7 | 1.0 | 0.50 | 0.70 | 0.21 |
| S2b | 4.90 | 9 | 0.8 | 0.65 | 0.85 | 0.18 |
| S3c | 4.70 | 7 | 0.7 | 0.50 | 1.05 | 0.11 |
aSand + substrate + coconut coir dust (1:1:1); bsand + substrate + coconut coir dust (3.5:3.5:2); csand + substrate (1:1)
Experimental setup
The experiment design was entirely randomized in a 3 × 3 factorial arrangement, with three inoculation treatments: seedlings inoculated with A. longula and G. albida and control plants (without inoculation) and three proportions of coconut coir dust (S1, S2, and S3), which are described above.
Plantlets inoculation and experimental conduction
The P. setacea seeds were granted by EMBRAPA — Cerrados via Technological Development for Functional and Medicinal use of Wild Passifloras — Passitec. To promote the germination, the seeds were scarified mechanically (Sandpaper no. 60), then two seeds were placed in pots (50 mL) with vermiculite and sand (Urimamã Mineração Ltda., Santa Maria da Boa Vista, Pernambuco, Brazil; Areiasil®, Sirinhaem, Pernambuco, Brazil) (1:1, v/v). After the emergence of leaves, the plantlets were transferred to pots containing the tested substrates and inoculated or not with soil-inoculum containing 300 glomerospores, hyphae, and roots colonized by A. longula and G. albida. The experiment was kept under environmental conditions (Tmín: 17 °C and Tmáx: 38.4 °C) and relative air humidity (AHmin: 67.9% and AHmax: 68.0%). After 68 days in a greenhouse, the plant material and soil were collected for analysis.
Mycorrhizae evaluations
The roots were washed in tap water, clarified with KOH (100 g L−1, w/v) (Fmaia®, Cotia, São Paulo, Brazil) and hydrogen peroxide (100 mL L−1, v/v) (Nuclear®, Diadema, São Paulo, Brazil), and stained with trypan blue (0.5 g L−1 in lactoglycerol, w/v) (Vetec®, Duque de Caxias, Rio de Janeiro, Brazil), as suggested by Phillips and Hayman [24]. The quadrants intersection method [25] was applied to measure the colonization percentage.
To extract the glomalin-related soil proteins (GRSP), 0.5 g of soil and 2 mL sodium citrate (20 mM) were added into test tubes (120 °C/30 min); subsequently, the material was centrifuged (10.000 rpm/10 min) and the easily extractable GRSP was at − 18 °C [26] to be quantified as total proteins [27].
Growth parameters
On the day of harvesting, the leaf area was measured by a handheld leaf area meter (CID Bio-Science, Cl 203, Camas, WA, US) and the dry matter of aerial parts was determined after drying in an oven (Quimis®, 0317 M-22, Diadema, São Paulo, Brazil, 60 °C) until reaching constant weight.
Spectrophotometric analyses
Extraction of biomolecules
The aerial part was cleaned and oven-dried (45 °C) (Quimis®, 0317 M-22, Diadema, São Paulo, Brazil) until stabilization of the phytomass (3 days). The leaves were cut (500 mg) and chemically macerated in 20 mL of ethanol (950 mL L−1, v/v) (Química moderna®, Barueri, São Paulo, Brazil) in amber flasks of 100 mL capacity for 12 days without stirring at 20 °C. Subsequently, the extracts were filtered (gauze and qualitative filter paper), transferred to flasks, and stored (− 18 °C) [20]. From these extracts, it was dosed total soluble carbohydrate, total proteins, total phenols, total tannins, total saponins, and the total antioxidant activity.
Biochemical analysis
In test tubes, the plant extract, distilled water, and phenol (800 mg L−1, W/v) (Vetec®, Duque de Caxias, Rio de Janeiro, Brazil) were added; the solution was vortex-stirred (Biomixer Ltda., VTX-2500, Morumbi, São Paulo, Brazil) and sulfuric acid was added (Química moderna®, Barueri, São Paulo, Brazil). The reading was performed at 490 nm (Thermo Scientific®, G105 UV–vis, Franklin, MA, USA), using glucose as the standard curve (y = 0.0006x − 0.0605; R2=0.9863) [28].
The plant extract and the Bradford reagent were added into test tubes and taken to the vortex (Biomixer Ltda., VTX-2500, Morumbi, São Paulo, Brazil) for 5 min. Afterward, the spectrophotometric reading at 595 nm was carried out. The bovine serum albumin (Sigma-Aldrich®, San Luis, MO, USA; Dinâmica®, São Paulo, Brazil) (y = 0.007x + 0.0036; R2=0.9972) [27] was used as a standard curve.
Phytochemical analysis
For the dosing of total phenols, the method of Folin–Ciocalteau was applied. The extract (200 μL), the Folin–Ciocalteau reagent (100 mL L−1, v/v) (Merck®, Darmstad, Hesse, Germany), and sodium carbonate (75 mg L−1, w/v) (Vetec®, Duque de Caxias, Rio de Janeiro, Brazil) were added in glass flasks; the solution was stirred in a vortex (Biomixer Ltda., VTX-2500, Morumbi, São Paulo, Brazil) and after 30 min, the reading was performed at 765 nm [29]. The tannic acid was used as the standard curve (Vetec, Duque de Caxias, Rio Janeiro, Brazil) (y = 7.9482x − 0.005; R2 = 0.9871).
The tannins quantification was carried out using the method of casein precipitation, as suggested by [30]. The solution was filtered and utilized on the total phenols [29]. The concentration of total tannins was obtained from the difference between the total phenolics concentration initially found in each sample and the value of the remaining total phenols after the casein precipitation.
The production of proanthocyanidins was evaluated by the vanillin acid method [31]. In glass flasks, the extract and acid vanillin (20 g L−1 w/v, in methanol) were added, and after 15 min of incubation, reading at 500 nm was carried out; the catechin (Sigma-Aldrich®, San Luis, MO, USA) was used for the standard curve construction (y = 0.412 × + 0.0026, R2 = 0.9999).
To quantify the production of saponins, the method of cobalt chloride was performed. In flasks, the extract, cobalt chloride (20 g L−1 w/v) (Nuclear®, Diadema, São Paulo, Brazil), and 1 mL of sulfuric acid (F Maia Ltda., Cotia, São Paulo, Brazil) were added [32]. The samples were stirred in a vortex (Biomixer Ltda., Morumbi, São Paulo, Brazil). The reading was carried out at 284 nm (Thermo Scientific®, G105 UV–vis, Franklin, MA, USA). The saponin (Inlab Ltda., São Luís, Maranhão, Brazil) was utilized for the standard curve (y = 0.0009x + 0.0045, R2 = 0.9926).
The total antioxidant activity was measured by the DPPH radical (2,2-diphenyl-picryl-hydrazyl) (Sigma-Aldrich®, San Luis, MO, USA). In amber flasks, the plant extract and DPPH solution (0.06 mM in methanol) (Sigma-Aldrich®, San Luis, MO, USA) were added, the samples were vortex-stirred (Biomixer Ltda., VTX-2500, Morumbi, São Paulo, Brazil), and after 30 min kept in the dark, a spectrophotometric reading (515 nm) was performed from the remaining DPPH in the solution [33]; the DPPH was used to obtain the standard curve (y = 0.0245 × 23 + 0.0033; R2 = 0.9999).
Statistical analyses
The data were submitted to ANOVA and the means were compared by Tukey’s test (5%) and it was performed on the Pearson correlation test (r) among the variables studied, using the Assistat program (7.7 Version).
Results and discussion
There was a highly significant interaction (p < 0.05) between the tested AMF and the substrates for all the evaluated parameters in P. setacea.
In S3, the passion fruit had an increase in the production of total saponins, when inoculated with G. albida which was 1,707.43% higher than the control plants (Table 2). Otherwise, the plants inoculated with A. longula did not obtain this increase (Table 2).
Table 2.
Total phenols concentration (mg g−1 plant), total tannins (mg g−1 plant), total proanthocyanidins (mg g−1 plant), total saponins (mg g−1 plant), and total antioxidant activity (mg of remaining DPPH−1 plant) in Passiflora setacea DC. leaves inoculated or not with Acaulospora longula Spain & N.C. Schenck and Gigaspora albida N.C. Schenck & G.S. Sm. and cultivated in soil with or without coconut coir dust addition, for 68 days in a greenhouse
| Inoculation treatments | Substrates | ||
|---|---|---|---|
| S1 Sand + substrate + coconut coir dust (1:1:1) |
S2 Sand + substrate + coconut coir dust (3.5:3.5:2) |
S3 Sand + substrate (1:1) |
|
| Total phenols | |||
| Control | 73.60 bB | 83.57 aB | 140.72 cA |
| A. longula | 87.06 aB | 55.04 bC | 240.91 bA |
| G. albida | 73.22 bB | 59.57 bC | 261.47 aA |
| CV%a | 6.14 | ||
| Total tannins | |||
| Control | 13.09 cB | 27.02 aA | 27.65 cA |
| A. longula | 31.45 bB | 7.55 bC | 136.12 bA |
| G. albida | 91.61 aB | 25.63 aC | 157.60 aA |
| CV%a | 6.57 | ||
| Total saponins | |||
| Control | 171.83 aA | 25.05 bB | 11.31 cB |
| A. longula | 17.83 cB | 18.18 bB | 44.98 bA |
| G. albida | 78.83 bB | 78.70 aB | 204.42 aA |
| CV%a | 12.30 | ||
| Total antioxidant activity | |||
| Control | 13.92 aA | 13.59 aA | 3.14 aB |
| A. longula | 13.76 aA | 13.87 aA | 2.09 aB |
| G. albida | 13.91 aA | 10.25 bB | 2.98 aC |
| CV%a | 9.69 | ||
| Total proanthocyanidins | |||
| Control | 4.98 aB | 10.24 aB | 30.66 aA |
| A. longula | 4.49 aB | 4.70 abB | 18.66 bA |
| G. albida | 4.75 aB | 3.56 bB | 17.92 bA |
| CV%a | 30.66 | ||
aCoefficient of variation. Means followed by the same letter, lower case in the column and upper case in the row, do not differ by Tukey’s test (5%)
In relation to the control without inoculation, the production of total tannins and of total phenols in plants associated with G. albida increased, respectively, 469.98% and 85.81% (Table 2); when associated with A. longula, the increment was, respectively, 392.30% and 71.20%, in relation to those non-inoculated and cultivated in S3 (Table 2).
In S3, the inoculated passion fruit increased the production of biomolecules, which are important for the herbal medicine industry, when compared to the non-inoculated plant, such as saponins, for those inoculated with G. albida (Table 2). The results reported in this study were greater than what was documented in yellow passion inoculated with G. albida (1.81 mg g−1 plant) [22].
Moreover, the increment of tannins and foliar phenols production in plants associated with G. albida was higher than in P. edulis inoculated with A. longula [18]. Similarly, in P. alata, it was documented the increase in the foliar total phenols content, when inoculated it was inoculated with Claroideoglomus etunicatum (W.N. Becker & Gerd.) C. Walker & A. Schüßler releasing the phosphate fertilization [34]. It is worth noting that this is the first report of the increase in the production of biomolecules, provided by mycorrhization in P. setacea.
The optimized production of phenolic compounds and saponins, registered in this study (Table 2), is relevant for passion fruit producers since they can use G. albida as a low-cost biotechnological alternative to the production of attractive phytomass to the anxiolytic herbal medicines industries. In this regard, for the multiplication and utilization of soil-inoculum with 300 glomerospores of this AMF, the cost is USD 0.12/plant, disregarding the installation investment for mycorrhizal inoculant production [35]. In addition, by dismissing the need for coconut coir dust supply in P. setacea cultivation, it can be saved USD 0.31 per seedling, as documented for the yellow passion fruit growing using the mycorrhizal technology [18].
In P. setacea colonized by G. albida, the total phenols concentration (Table 2) was higher than those obtained from foliar hydroalcoholic extracts of P. setacea collected in the caatinga (22.17 mg g−1 plant) [36], reinforcing the importance of this technology applied to optimize the compounds of the phytomass of this plant by the producers.
Furthermore, the plants cultivated in S3 had the highest mycorrhizal colonization percentage when inoculated with G. albida (49%) in comparison to those without AMF inoculation (27.81%), contrasting to plants colonized by A. longula, which had 28.63% of colonization percentage (Table 3). The colonization rate was correlated (p < 0.01) with all metabolites and the antioxidant activity (Table 4). Moreover, when P. setacea seedlings were inoculated with G. albida, the soil was benefited from GRSP concentration in S1 or S3 (Table 3). Furthermore, the production of phenolic compounds was positively correlated with the production of these proteins (r = 0.5015; p < 0.01) (Table 4). This benefit occurred in a lesser proportion in plants inoculated with A. longula and grown in S1 with an increment of 17.10% in comparison to control plants (Table 3).
Table 3.
Concentration of glomalin-related soil proteins (GRSP) (mg g−1 soil) and mycorrhizal colonization (%) of Passiflora setacea DC. seedlings inoculated or not with Acaulospora longula Spain & N.C. Schenck and Gigaspora albida N.C. Schenck & G.S. Sm. and cultivated in soil with or without coconut coir dust addition, for 68 days in a greenhouse
| Inoculation treatments | Substrates | ||
|---|---|---|---|
| S1 Sand + substrate + coconut coir dust (1:1:1) |
S2 Sand + substrate + coconut coir dust (3.5:3.5:2) |
S3 Sand + substrate (1:1) |
|
| Mycorrhizal colonization (%) | |||
| Control | 30.00 aA | 27.12 aA | 27.81 bA |
| A. longula | 8.50 bB | 24.50 aA | 28.63 bA |
| G. albida | 16.00 bC | 26.50 aB | 49.00 aA |
| CV%a | 22.33 | ||
| GRSP (mg g−1 soil) | |||
| Control | 0.76 cB | 0.91 aA | 0.93 bA |
| A. longula | 0.89 bA | 0.89 aA | 0.90 bA |
| G. albida | 0.98 aB | 0.90 aC | 1.06 aA |
| CV%a | 3.61 | ||
aCoefficient of variation. Means followed by the same letter, lower case in the column and upper case in the row, do not differ by Tukey’s test (5%)
Table 4.
Pearson’s correlation (r) between leaf area (LA), dry matter from aerial parts (DMAP), mycorrhizal colonization (MC), total phenols, total tannins, total proanthocyanidins (Proant.), total saponins, total antioxidant activity (AOA), total soluble carbohydrates (Carbo.), total proteins, and glomalin-related soil proteins (GSRP) in Passiflora setacea DC. inoculated or not with arbuscular mycorrhizal fungi and cultivated or not in substrate containing coconut coir dust
| LA | DMAP | MC | Phenols | Tannins | Proant | Saponins | AOA | Carbo | Proteins | GSRP | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| LA | - | 0.9083** | 0.5181** | 0.8664** | 0.6075** | 0.8893** | ns | − 0.9560** | 0.9448** | ns | 0.4269** |
| DMAP | - | 0.5049** | 0.8946** | 0.7123** | 0.7104** | ns | − 0.9406** | 0.8105** | ns | 0.3512* | |
| MC | - | 0.5701** | 0.4351** | 0.3342** | 0.5815** | − 0.5545** | 0.3497* | ns | ns | ||
| Phenols | - | 0.8617** | 0.6353** | 0.3578* | − 0.8555** | 0.7312** | ns | 0.5015** | |||
| Tannins | - | 0.3360* | 0.4378** | − 0.6363** | 0.3761* | ns | 0.6358** | ||||
| Proant | - | ns | − 0.7974** | 0.9137** | ns | 0.3849* | |||||
| Saponins | - | ns | ns | ns | ns | ||||||
| AOA | - | − 0.8584** | ns | − 0.4307** | |||||||
| Carbo | - | ns | ns | ||||||||
| Proteins | - | ns | |||||||||
| GSRP | - |
** p < 0.01; * p < 0.05; ns, non-significant
It is important to highlight that the plants cultivated in S3 had a higher mycorrhizal colonization percentage when inoculated with G. albida and A. longula in relation to the non-inoculated seedlings (Table 3). It differs from the behavior registered in another passion fruit species colonized by A. longula [18]. The greater mycorrhizal colonization by G. albida may favor the accumulation of secondary compounds in this study, as suggested in a study with P. alata [34]. One of the possible explanations is that mycorrhization can regulate P transporters and improve the absorption of nutrients [37], which is important in phenolic anabolic routes and participates in the shikimic acid phosphorylation [38].
On the other hand, the reduced colonization percentage in P. setacea roots cultivated in S1 may have occurred due to the presence of some compound present in this soil conditioner and may have limited the AMF development in this case; complementary studies are needed to prove this hypothesis. Moreover, the substrate pH with the coconut coir dust may have disfavored the colonization, considering that A. longula and some Gigasporaceae occur in acid soils [39].
The G. albida benefit to increase the production of GRSP in S3 (Table 3) and the positive correlation of these proteins’ production and the concentration of phenolic compounds in P. setacea is a fact that has not been documented yet; the longer mycelial extension, that is a Gigasporaceae characteristic [40], may have enabled the increased GRSP production, which is important to organic matter stabilization [41] and to nutrient mineralization by microorganism action [42]. Similar results were observed in the cultivation of Physalis peruviana L., in which the GRSP production was favored by mycorrhization with Claroideoglomus claroideum (N.C. Schenck & G.S. Sm.) C. Walker & A. Schüßler [43].
On the other hand, the production of foliar proanthocyanidins and foliar proteins was not benefited by the mycorrhization, with the highest concentration of these biomolecules verified in non-inoculated plants and cultivated in S3 (Tables 2 and 5). In a different way, the concentration of total soluble carbohydrates was reduced by 60.81% in plants inoculated with G. albida in S3, in relation to the control without inoculation; this pattern did not occur in plants colonized by A. longula and cultivates in S1 and S2, since no effect was seen (Table 5). In the S3, the greatest concentration of secondary metabolites was documented (Table 2). It is worth noting the positive correlation between leaf area and the carbohydrates concentration (r = 0.9448; p < 0.01) (Table 4). Similarly, the mycorrhization did not optimize the antioxidant activity in P. setacea extracts apart from the plants cultivated in S2 and colonized by G. albida (Table 2).
Table 5.
Concentration of total soluble carbohydrates (mg g−1 plant) and total proteins (mg g−1 plant) in Passiflora setacea DC. leaves inoculated or not with Acaulospora longula Spain & N.C. Schenck and Gigaspora albida N.C. Schenck & G.S. Sm. and cultivated in soil with or without coconut coir dust addition, for 68 days in a greenhouse
| Inoculation treatments | Substrates | ||
|---|---|---|---|
| S1 Sand + substrate + coconut coir dust (1:1:1) |
S2 Sand + substrate + coconut coir dust (3.5:3.5:2) |
S3 Sand + substrate (1:1) |
|
| Total soluble carbohydrates | |||
| Control | 113.99 aC | 160.08 aB | 620.72 aA |
| A. longula | 174.71 aB | 132.28 aC | 474.47 bA |
| G. albida | 34.97 bC | 56.22 bB | 377.45 cA |
| CV%a | 8.21 | ||
| Total proteins | |||
| Control | 342.97 bC | 1528.72 aA | 889.57 aB |
| A. longula | 448.15 bB | 648.97 bA | 613.70 bA |
| G. albida | 649.22 aA | 537.72 cB | 695.80 bA |
| CV%a | 8.83 | ||
aCoefficient of variation. Means followed by the same letter, lower case in the column and upper case in the row, do not differ by Tukey’s test (5%)
The reduction of total soluble carbohydrates production can be related to the targeting of metabolic intermediates, such as erythrose-4-phosphate and phosphoenolpyruvate, pentose, and glycolytic pathway products, to the shikimic acid synthesis, the main precursor of the phenolic compounds route [38].
Otherwise, previous research with P. edulis documented that AMF inoculation associated with coconut coir dust or vermicompost favored foliar carbohydrates and proteins’ production [18]. However, in Punica granatum L. inoculated with A. longula and cultivated in a substrate with vermicompost, the reduction of carbohydrates production was observed [35]. It is important to develop studies with longer experimentation time and test other fertilizers, since the production of these biomolecules can vary in accordance with several factors, such as the physical–chemical characteristics of the substrate [18, 22]. Moreover, it is possible that the distinguished combination of the substrates can promote its benefits along with an efficient AMF [44].
Concerning the proanthocyanidins accumulation, it is likely that non-mycorrhizal P. setacea seedlings, cultivated in S3, were greater due to the targeting of some phenolic compounds to the proanthocyanidins biosynthesis, opposing to previous research [45], considering that plants of this same treatment presented reduction of total phenols production (Table 2). Similar results were observed in Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz, since the mycorrhization did not favor the production of these biomolecules in the fruits [46].
Despite the production of total proteins not being influenced by mycorrhization, in plants cultivated in S2 and S3, and not correlated with all the studied parameters (Table 4), these biomolecules are a source of aromatic amino acids, such as phenylalanine, that can be relevant for phenolics synthesis [38]. Similarly, the Mimosa tenuiflora (Wild.) Poir. mycorrhization did not favor the production of total proteins [47].
Furthermore, the in vitro antioxidant activity of P. setacea was negatively correlated with some secondary metabolites (Table 4). These results differed from what was documented in mycorrhizal strawberry with Claroideoglomus claroideum (N.C. Schenck & G.S. Sm.) C. Walker & A. Schüßler and manure as fertilizer [16]. For a better understanding of the mycorrhizal symbiosis effect in the P. setacea antioxidant potential, other radicals (TPTZ and ABTS) should be tested in future research.
Regarding the growth parameters, the application of A. longula favored the accumulation of dry matter of the plants, dispensing with the incorporation of coconut coir dust into the seedling substrate, unverified benefit to the leaf area (Table 6). In this context, the larger area extension for light absorption can have intensified the photosynthesis, as verified in cotton [48], which did not occur in plants grown in S3 (Table 4). In contrast to the registered in our study, in sweet passion fruit, the increment in the leaf area, in the dry matter from aerial parts and the stem diameter occurred in mycorrhizal plants that were cultivated in vermicompost, a fertilizer rich in nutrients [20].
Table 6.
Leaf area (cm2) and dry matter of aerial parts (DMAP) (g) of Passiflora setacea DC. seedlings inoculated or not with Acaulospora longula Spain & N.C. Schenck and Gigaspora albida N.C. Schenck & G.S. Sm. and cultivated in soil with or without coconut coir dust addition, for 68 days in a greenhouse
| Inoculation treatments | Substrates | ||
|---|---|---|---|
| S1 Sand + substrate + coconut coir dust (1:1:1) |
S2 Sand + substrate + coconut coir dust (3.5:3.5:2) |
S3 Sand + substrate (1:1) |
|
| Leaf area (cm2) | |||
| Control | 1.26 aC | 4.35 aB | 94.08 aA |
| A. longula | 0.78 aB | 2.74 abB | 82.93 bA |
| G. albida | 0.71 aB | 1.30 bB | 83.07 bA |
| CV%a | 4.8 | ||
| DMAP (g) | |||
| Control | 0.10 aB | 0.10 bB | 0.36 cA |
| A. longula | 0.10 aB | 0.13 abB | 0.54 aA |
| G. albida | 0.10 aC | 0.15 aB | 0.39 bA |
| CV%a | 13.05 | ||
aCoefficient of variation. Means followed by the same letter, lower case in the column and upper case in the row, do not differ by Tukey’s test (5%)
The mycorrhization benefit for some allometric parameters in seedlings grown in non-fertilized soils contrasts with the results found in P. setacea inoculated with C. etunicatum and had greater growth regardless of phosphate fertilization [19]. In this experiment, to increase the dry matter from the aerial parts, the mycorrhization was sufficient, reducing costs with coconut coir dust supplementation (Table 6). This result differs from the obtained in Crocus sativus L. grown in vermicompost and inoculated with AMF since the interaction between these factors enhanced the plant growth [49].
In this study, the hypothesis was not corroborated, since the cultivation of mycorrhizal P. setacea dismisses the coconut coir dust addition when they are in AMF symbiosis, especially applying G. albida as inoculant that has the greatest physiological compatibility with this Passiflora species. By that, the application of this AMF is sufficient to increase the phytochemical production, plant growth, colonization, and mycorrhizal activity, measured by GRSP content in the soil.
In conclusion, the G. albida inoculation in P. setacea is an alternative to provide phytomass with higher phenolic compounds concentration and saponins of interest to the anxiolytic herbal medicine industries based on Passiflora.
Acknowledgements
This research was supported by the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES) — Finance Code 001. The authors would like to thank Embrapa Cerrados (Brazil) for donating Passiflora setacea seeds.
Author contribution
Conceptualization: Silva, F.S.B.; data curation: Muniz, B.C., Silva, F.S.B.; formal analysis: Muniz, B.C., Silva, F.S.B.; funding acquisition: Silva, F.S.B.; investigation: Muniz, B.C., Falcão, E.L.; methodology: Muniz, B.C., Falcão, E.L., Silva, F.S.B.; project administration: Silva, F.S.B.; resources: Silva, F.S.B.; supervision: Silva, F.S.B.; visualization: Muniz, B.C., Falcão, E.L., Silva, F.S.B.; writing — original draft preparation: Muniz, B.C., Falcão, E.L., Silva, F.S.B.; writing — review and editing: Muniz, B.C., Falcão, E.L., Silva, F.S.B.
Funding
This research was supported by the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES) — Finance Code 001.
Data availability
Not applicable.
Declarations
Ethics approval
Not applicable.
Consent
All authors consent the publication of this research.
Conflict of interest
The authors declare no competing interests.
Footnotes
Responsible Editor: Jerri Zilli
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