The Open Microbiology Journal




ISSN: 1874-2858 ― Volume 13, 2019
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Arbuscular Mycorrhizal Fungi Increase the Phenolic Compounds Concentration in the Bark of the Stem of Libidibia Ferrea in Field Conditions



Emanuela Lima dos Santos1, 2, 3, *, Francineyde Alves da Silva2, Fábio Sérgio Barbosa da Silva1, 2, 3
1 Post-graduation in Cellular and Applied Molecular Biology, Institute of Biological Sciences, University of Pernambuco, 310, Arnóbio Marques Street, Santo Amaro – 50100130 - Recife, Brazil
2 University of Pernambuco, campus Petrolina, Laboratory of Mycorrhizal Technology (LTM/UPE)- Petrolina Center, BR 203, Km 2, 56328-900-Petrolina, Brazil
3 University of Pernambuco, campus Santo Amaro, Laboratory of Mycorrhizal Technology (LTM/UPE) – Recife Center, 310, Arnóbio Marques Street, Santo Amaro – 50100130 - Recife, Brazil

Abstract

Background:

Libidibia ferrea is a species particular to the caatinga presenting medicinal properties for containing bioactive compounds. The use of Arbuscular Mycorrhizal Fungi (AMF) can increase the production of biomolecules in the legume leaves; however, no light has been shed on the role of symbiosis in maximizing metabolites production in the bark of L. ferrea stem.

Objective:

The aim was to select AMF that are efficient at increasing the production of phenolic compounds with medicinal properties in the bark of the L. ferrea stem.

Methods:

The experiment was designed in randomized blocks with four inoculation treatments (plants pre-inoculated with Claroideoglomus etunicatum, with Gigaspora albida, with Acaulospora longula, and non-inoculated plants – control) with six repetitions. Thirteen months after the transplanting, the plants were pruned and the bark of the stem was collected; subsequently, this plant material was dried in a chamber. After the drying process, fractions of the bark of the stem were macerated in methanol. The extracts were further used for analyses of the biomolecules.

Results:

The flavonoids concentration had an increase of, respectively, 236% and 186% in relation to the control for the treatments with A. longula and C. etunicatum; plants inoculated with A. longula had an increase of 47% in total tannins concentration compared with the non-inoculated control – a benefit that the proanthocyanidins did not present.

Conclusion:

Applying inoculation with A. longula may be an alternative to increase the production of biomolecules of the secondary metabolism in the bark of the L. ferrea stem in field conditions.

Keywords: Caatinga, AMF, Bioactive compounds, Glomeromycota, Secondary metabolism, Field conditions.


Article Information


Identifiers and Pagination:

Year: 2017
Volume: 11
First Page: 283
Last Page: 291
Publisher Id: TOMICROJ-11-283
DOI: 10.2174/1874285801711010283

Article History:

Received Date: 11/07/2017
Revision Received Date: 06/10/2017
Acceptance Date: 07/10/2017
Electronic publication date: 31/10/2017
Collection year: 2017

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© 2017 Santos et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Post-graduation in Cellular and Applied Molecular Biology, Institute of Biological Sciences, University of Pernambuco, 310, Arnóbio Marques Street, Santo Amaro – 50100130 - Recife, PE; University of Pernambuco, campus Petrolina, Laboratory of Mycorrhizal Technology (LTM/UPE)- Petrolina Center, BR 203, Km 2, 56328-900-Petrolina, PE; University of Pernambuco, campus Santo Amaro, Laboratory of Mycorrhizal Technology (LTM/UPE) – Recife Center, 310, Arnóbio Marques Street, Santo Amaro – 50100130 - Recife, PE- Brazil; Tel: 55+813183-3316; E-mails: emanuela_lima07@hotmail.com, emanuela_lima@oi.com.br




1. INTRODUCTION

Libidibia ferrea is a medicinal tree legume particular to the caatinga, popularly known as pau-ferro or jucá [1Maia-Silva C, Silva CI, Hrncir M, Queiroz RT, Imperatriz-Fonseca VL. Árvores. Guia de plantas visitadas por abelhas na Caatinga Fortaleza 2012.]. The population uses it in the treatment of diabetes, anemia, respiratory and gastrointestinal diseases [2de Albuquerque UP, Monteiro JM, Ramos MA, de Amorim EL. Medicinal and magic plants from a public market in northeastern Brazil. J Ethnopharmacol 2007; 110(1): 76-91.
[http://dx.doi.org/10.1016/j.jep.2006.09.010] [PMID: 17056216]
] because of its therapeutic properties such as anti-inflammatory [3Pereira LdeP, da Silva RO, Bringel PH, da Silva KE, Assreuy AM, Pereira MG. Polysaccharide fractions of Caesalpinia ferrea pods: potential anti-inflammatory usage. J Ethnopharmacol 2012; 139(2): 642-8.
[http://dx.doi.org/10.1016/j.jep.2011.12.012] [PMID: 22178173]
], antimicrobial [4de Oliveira Marreiro R, Bandeira MF, de Souza TP, et al. Evaluation of the stability and antimicrobial activity of an ethanolic extract of Libidibia ferrea. Clin Cosmet Investig Dent 2014; 6: 9-13.
[PMID: 24501546]
], among others.

The medicinal action of L. ferrea is associated with the presence of secondary compounds in the phytomass found in different parts of the plant, such as flavonoids, phenols, tannins, and others [5Silva FA, Silva FS, Maia LC. Biotechnical application of arbuscular mycorrhizal fungi used in the production of foliar biomolecules in ironwood seedlings. J Med Plants Res 2014; 8: 814-9. a [Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz var. ferrea].
[http://dx.doi.org/10.5897/JMPR2014.5358]
]. Because of its therapeutic potential, L. ferrea is included in the Relação Nacional de Plantas Medicinais de Interesse ao Sistema Único de Saúde (Renisus - Brazil) [6Saúde M. Relação de Plantas Medicinais de Interesse ao SUS 2009.].

The optimization in the production of these biomolecules may be achieved through the inoculation of Arbuscular Mycorrhizal Fungi (AMF) [5Silva FA, Silva FS, Maia LC. Biotechnical application of arbuscular mycorrhizal fungi used in the production of foliar biomolecules in ironwood seedlings. J Med Plants Res 2014; 8: 814-9. a [Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz var. ferrea].
[http://dx.doi.org/10.5897/JMPR2014.5358]
, 7Hazzoumi Z, Moustakime Y, Elharchli EH, Joutei KA. Effect of arbuscular mycorrhizal fung[i (AMF) and water stress on growth, phenolic compounds, glandular hairs, and yield of essential oil in basil (Ocimum gratissimum L). Chem Biol Technol Agric 2015; 2: 2-11.
[http://dx.doi.org/10.1186/s40538-015-0035-3]
, 8Oliveira PT, Alves GD, Silva FA, Silva FS. Foliar bioactive compounds in Amburana cearensis (Allemao) A. C. Smith seedlings : Increase of biosynthesis using mycorrhizal technology. J Med Plants Res 2015; 9: 712-8.
[http://dx.doi.org/10.5897/JMPR2015.5798]
]. AMF are microorganisms belonging to phylum Glomeromycota [9Schübler A, Schwarzott D, Walker C. A new fungal phylum, the Glomeromycota: phylogeny and evolution. Mycol Res 2001; 105: 1413-21.
[http://dx.doi.org/10.1017/S0953756201005196]
] that form mutualistic symbiosis with the plants and colonize most of the studied angiosperms and gymnosperms, in addition to some pteridophytes and bryophytes [10Smith SE, Read DJ. Mycorrhizal symbiosis 3rd ed. 2008.].

When in symbiosis, the AMF improve the nutritional condition of plants [11Selvaraj T, Nisha MC, Rajeshkumar S. Effect of indigenous arbuscular mycorrhizal fungi on some growth parameters and phytochemical constituents of Pogostemon patchouli Pellet. J Sci Technol 2009; 3: 222-34., 12Karagiannidis N, Thomidis T, Lazari D, Panou-filotheou E, Karagiannidou C. Effect of three Greek arbuscular mycorrhizal fungi in improving the growth, nutrient concentration, and production of essential oils of oregano and mint plants. Sci Hortic (Amsterdam) 2011; 129: 329-34.
[http://dx.doi.org/10.1016/j.scienta.2011.03.043]
], promote their development [13Freitas MS, Martins MA, Carvalho AJ, Carneiro RF. Crescimento e produção de fenóis totais em carqueja [Baccharis trimera (Less.) DC.] na presença e na ausência de adubação mineral. Rev Bras Pl Med 2004; 6: 30-4.], and increase the production of secondary compounds adding value to the plant phytomass, which may improve its therapeutic potential [14Oliveira MS, Campos MA, Albuquerque UP, Silva FS. Arbuscular Mycorrhizal Fungi (AMF) affects biomolecules content in Myracrodruon urundeuva seedlings. Ind Crops Prod 2013; 50: 244-7.
[http://dx.doi.org/10.1016/j.indcrop.2013.07.041]
-17Zitter-Eglseer K, Nell M, Lamien-Meda A, Steinkellner S, Wawrosh C, Kopp B, et al. Effects of root colonization by symbiotic arbuscular mycorrhizal fungi on the yield of pharmacologically active compounds in Angelica archangelica L. Acta Physiol Plant 2015; 37: 2-11.].

Some mechanisms are suggested to explain the accumulation of compounds in the secondary plant metabolism in response to mycorrhizal symbiosis as an improvement in the nutritional condition of the host [18Zimare SB, Borde MY, Jite PK, Malpathak NP. Effect of AM Fungi (Gf, Gm) on Biomass and Gymnemic Acid Content of Gymnema sylvestre (Retz.) R. Br. ex Sm. Proc Natl Acad Sci, India, Sect B Biol Sci 2013; 83: 439-45.
[http://dx.doi.org/10.1007/s40011-013-0159-9]
, 19Riter Netto AF, Freitas MS, Martins MA, Carvalho AJ, Vitorazi Filho JÁ. Efeito de fungos micorrízicos arbusculares na bioprodução de fenóis totais e no crescimento de Passiflora alata Curtis. Rev Bras Pl Med 2014; 16: 1-9.
[http://dx.doi.org/10.1590/S1516-05722014000100001]
] as well as alterations in the activity of key-enzymes [20Zhang RQ, Zhu HH, Zhao HQ, Yao Q. Arbuscular mycorrhizal fungal inoculation increases phenolic synthesis in clover roots via hydrogen peroxide, salicylic acid and nitric oxide signaling pathways. J Plant Physiol 2013; 170(1): 74-9.
[http://dx.doi.org/10.1016/j.jplph.2012.08.022] [PMID: 23122788]
], activation of metabolic routes [21Lohse S, Schliemann W, Ammer C, Kopka J, Strack D, Fester T. Organization and metabolism of plastids and mitochondria in arbuscular mycorrhizal roots of Medicago truncatula. Plant Physiol 2005; 139(1): 329-40.
[http://dx.doi.org/10.1104/pp.105.061457] [PMID: 16126866]
], increase in gene expression [21Lohse S, Schliemann W, Ammer C, Kopka J, Strack D, Fester T. Organization and metabolism of plastids and mitochondria in arbuscular mycorrhizal roots of Medicago truncatula. Plant Physiol 2005; 139(1): 329-40.
[http://dx.doi.org/10.1104/pp.105.061457] [PMID: 16126866]
, 22Mandal S, Upadhyay S, Singh VP, Kapoor R. Enhanced production of steviol glycosides in mycorrhizal plants: a concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes. Plant Physiol Biochem 2015; 89: 100-6.
[http://dx.doi.org/10.1016/j.plaphy.2015.02.010] [PMID: 25734328]
], among others.

Few are the studies associating the production of secondary metabolites with inoculated plants established in the field [23Gupta ML, Prasad A, Ram M, Kumar S. Effect of the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum on the essential oil yield related characters and nutrient acquisition in the crops of different cultivars of menthol mint (Mentha arvensis) under field conditions. Bioresour Technol 2002; 81(1): 77-9.
[http://dx.doi.org/10.1016/S0960-8524(01)00109-2] [PMID: 11708758]
-26Kara Z, Arslan D, Güler M, Güler S. Inoculation of arbuscular mycorrhizal fungi and application of micronized calcite to olive plant: Effects on some biochemical constituents of olive fruit and oil. Sci Hortic (Amsterdam) 2015; 185: 219-27.
[http://dx.doi.org/10.1016/j.scienta.2015.02.001]
]; among the existing studies, only one was conducted in Brazil, with Silva et al. [27Silva FA, Ferreira MR, Soares LA, Sampaio EV, Silva FS, Maia LC. Arbuscular mycorrhizal fungi increase gallic acid production in leaves of field grown Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz. J Med Plants Res 2014; 8: 1110-5. b
[http://dx.doi.org/10.5897/JMPR2013.5503]
] recording an increase in the foliar concentration of Gallic acid in mycorrhizal L. ferrea.

There are no records on the production of bioactive compounds in the bark of the L. ferrea stem in response to mycorrhizal inoculation. Considering that such part of the plant is highly used as therapeutic alternative, the aim of this study was to select AMF that are efficient at increasing the production of phenolic compounds with medicinal properties in the bark of the L. ferrea stem; in this context, we tested the hypothesis that inoculation with AMF increases the production of phenolic compounds in the bark of the L. ferrea stem; however, the benefits vary according to the inoculated AMF.

2. MATERIAL AND METHODS

We developed the experiment in February, 2013 at the Experimental Field of the Mycorrhizal Technology Laboratory (LTM/UPE), located at University of Pernambuco, campus Petrolina, BR 203, Km 2, Petrolina, PE- Brazil. The phytochemical and mycorrhizal assessments were carried out 13 months after the field transplanting.

2.1. Experimental Design

We conducted the experimental design in randomized blocks with four inoculation treatments (plants pre-inoculated with C. etunicatum, plants pre-inoculated with G. albida, plants pre-inoculated with A. longula, and non-inoculated plants – control) with six repetitions.

2.2. Arbuscular Mycorrhizal Fungi

We employed three AMF isolates: Acaulospora longula Spain & N.C. Schenck (UFPE 21), Claroideoglomus etunicatum (W. N. Becker & Gerdemann) C. Walker & A.Schussler) (UFPE 06), and Gigaspora albida N.C. Schenck & G.S. Sm. (UFPE 01) provided by the Mycorrhizae Laboratory of the Mycology Department of the Federal University of Pernambuco. The AMF were multiplied on soil with ten percent of vermicompost and Panicum miliaceum L. as the host; the inoculum was stored at 4 ºC until use.

2.3. Production of the L. ferrea Seedlings

We produced the L. ferrea seedlings using experimental screen, covering the period between July 2012 and February 2013. The seeds were germinated and transferred to pots with soil containing 1.2 Kg soil + five percent vermicompost when the plantlets presented two definitive leaves and subsequently inoculated or not with soil inoculum containing 200 spores, hyphae, and colonized roots from each AMF tested. After 225 days, the seedlings were transplanted into the field. At the transplanting, the seedlings presented in average 72 cm of height, 5.1 mm of stem diameter and 14 leaves as well as a colonization rate varying from 6.2% for the control plants to 53.45% for the treatments of inoculation.

2.4. Preparing the Experimental Area

Before the installation of the experiment, the area with 2,400 m2 was ploughed, gridded, and caved. Each cave (40 x 40 x 40 cm) was fertilized using five liters of vermicompost and 150 g of simple superphosphate; the irrigation was carried out using semi-automatic dripping (8.4 L H2O plant-1 h-1). The soil of the experimental field presented the following chemical characteristics at the depth of 0 – 20 cm: P 10.38 mg dm -3, K 0.24 cmolc dm-3, Ca 1.4 cmolc dm-3, Mg 0.5 cmolc dm-3, Na 0.03 cmolc dm-3, Al 0.00 cmolc dm-3, organic matter 0.41 g Kg-1, pH 6.2, electrical conductivity 0.21 mS cm-1.We transplanted 96 plants arranged in six blocks, consisted of four plants per treatment, considering two plants for analyses, resulting 16 plants per block. The plants were arranged in lines with a border around the experimental field with non-mycorrhizal L. ferrea.

2.5. Phytochemical Analyses

2.5.1. Preparing the Plant Extract

Thirteen months after the beginning of the experiment, we pruned the plants and collected the bark of the stem; subsequently, we dried this plant material in a chamber (Biopar, Porto Alegre, RS, Brazil) (45 ºC) for three days. After the drying process, fractions of 500 mg of the bark of the stem were macerated in 20 mL methanol (70%, v/v) (F Maia, Cotia, Brazil) for ten days at 20 ºC [28Brito HO, Noronha EP, França LM, Brito LM, Prado MA. Análise da composição fitoquímica do extrato etanólico das folhas da Annona squamosa (ATA). Rev Bras Farmacogn 2008; 89: 180-4.]. The extracts were gauze-filtered, re-filtered in qualitative filter paper, and stored in amber flasks in a freezer.

2.5.2. Flavonoids

For the quantification of flavonoids were transferred to a volumetric flask (25 mL): 1 mL methanolic extract, 0.6 mL glacial acetic acid (F Maia, Cotia, Brazil) and added 10 mL of a pyridine/methanol solution (2:8, v/v) (Vetec, Duque de Caxias, Brazil/ F Maia, Cotia, Brazil) and 2.5 mL aluminum chloride (5%, w/v) (Vetec, Duque de Caxias, Brazil) in methanol (F Maia, Cotia, Brazil). The volume was completed with distilled water and the solution was put to rest for 30 minutes; subsequently, we carried out a reading using a spectrophotometer (Biospectro, Curitiba, Brazil) (420 nm) with rutin (Sigma-Aldrich, São Paulo, Brazil) in the standard curve [29de Sousa Araújo TA, Alencar NL, de Amorim EL, de Albuquerque UP. A new approach to study medicinal plants with tannins and flavonoids contents from the local knowledge. J Ethnopharmacol 2008; 120(1): 72-80.
[http://dx.doi.org/10.1016/j.jep.2008.07.032] [PMID: 18725282]
].

2.5.3. Total Phenols

Total phenols were quantified according to the Folin-Ciocalteau method: 2 mL of the extract in volumetric flask (100 mL) added with five mL of the Folin-Ciocalteau reagent (10%, v/v) (Merck, Rio de Janeiro, Brazil) and 10 mL of sodium carbonate solution (7.5%, w/v) (F Maia, Cotia, Brazil). The volume was completed with distilled water and the solution remained at rest for 30 minutes. Subsequently, we conducted a spectrophotometric reading (760 nm) using tannic acid (Vetec, Duque de Caxias, Brazil) for the standard curve [30Monteiro JM, Albuquerque UP, Neto EM, Araújo EL, Albuquerque MM, Amorim EL. The effects of seasonal climate changes in the Caatinga on tannin levels in Myracrodruon urundeuva (Engl.) Fr. All. and Anadenanthera colubrina (Vell.) Brenan. Rev Bras Farmacogn 2006; 16: 338-44.
[http://dx.doi.org/10.1590/S0102-695X2006000300010]
].

2.5.4. Total Tannins

We carried out the quantification using modified casein precipitation method. We introduced three mL of the extract in an amber flask added with 0.5 g casein (Vetec, Duque de Caxias, Brazil); subsequently, the mixture was stirred for three hours (160 rpm) at 25 ºC. The samples passed through qualitative filter paper, the volume was inserted in a volumetric flask and completed until 25 mL with distilled water. We carried out the quantification of phenols for this solution using the Folin-Ciocalteau method [30Monteiro JM, Albuquerque UP, Neto EM, Araújo EL, Albuquerque MM, Amorim EL. The effects of seasonal climate changes in the Caatinga on tannin levels in Myracrodruon urundeuva (Engl.) Fr. All. and Anadenanthera colubrina (Vell.) Brenan. Rev Bras Farmacogn 2006; 16: 338-44.
[http://dx.doi.org/10.1590/S0102-695X2006000300010]
]. The concentration of total tannins was obtained from the comparison of the difference of the values found in this analysis and the values found in the analysis of total phenols. We used tannic acid as standard curve (Vetec, Duque de Caxias, Brazil).

2.5.5. Total Proanthocyanidins

Total proanthocyanidins were dosed through modified acid-vanillin method [31Queiroz CR. Morais SAL, Nascimento EA. Caracterização dos taninos da aroeira-preta (Myracrodruon urundeuva). Rev Arvore 2002; 26: 485-92.
[http://dx.doi.org/10.1590/S0100-67622002000400011]
]. We introduced 10 µL of the extract in a test tube and 990 µL distilled water to dilute the extract as well as 2 mL vanillin solution (2%, w/v) (Vetec, Duque de Caxias, Brazil) in H2SO4 (70%, v/v) (F Maia, Cotia, Brazil). The solution remained at rest in the dark for 15 minutes; subsequently, we carried out a spectrophotometric reading (500 nm) using calibration curve prepared with catechin solution.

2.6. Mycorrhizal Analyses

2.6.1. Mycorrhizal Colonization and Spores Density

For the mycorrhizal analyses, we collected soil at three equidistant points in the rhizosphere of the plants with depth between zero and 20 cm. For the mycorrhizal colonization, the roots were clarified with KOH (10%, w/v) and hydrogen peroxide (10%, v/v) (F Maia, Cotia, Brazil) as well as colored with Trypan blue (0.05 w/v, in lactoglycerol) (Vetec, Duque de Caxias, Brazil) according to method by Philips and Hayman [32Phillips JM, Hayman DS. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br Mycol Soc Br Mycol Soc 1970; 55: 158-61.
[http://dx.doi.org/10.1016/S0007-1536(70)80110-3]
]. We estimated the colonization percentage through the quarter intersect method [33Giovannetti M, Mosse B. An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol 1980; 84: 489-500.
[http://dx.doi.org/10.1111/j.1469-8137.1980.tb04556.x]
]. The glomerospores were extracted from the soil using the wet sieving methodology, decantation [34Gerdemann JW, Nicolson TH. Spores of mycorrhizal endogone species extracted from soil by wet sieving and decanting. Trans Br Mycol Soc 1963; 46: 235-44.
[http://dx.doi.org/10.1016/S0007-1536(63)80079-0]
], and centrifugation in water and sucrose (45%, w/v) proposed by Jenkins [35Jenkins WR. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Dis Res 1964; 48: 692.] and quantified in stereomicrocospe (40 x)

2.6.2. Statistical Analysis

The data were subjected to ANOVA and the means compared using Tukey test (P < 0.05) with Assistat software (7.7).

3. RESULTS

We recorded an increase of 30% in the diameter of the root for the plants inoculated with A. longula and 35% for those associated with G. albida in relation to non-inoculated control (Table 1).

The rate of mycorrhizal colonization did not differ among the treatments (Table 1). A higher density of spores was recorded in the rhizosphere of inoculated plants with emphasis to the treatments with A. longula and C. etunicatum, presenting the highest densities (Table 1).

Averages followed by the same letter do not differ from the Tukey test (P < 0.05).


Table 1
Stem diameter, colonization, spore density, in Libidibia ferrea plants, in the field, inoculated or non-inoculated with arbuscular mycorrhizal fungi (AMF), 13 months after transplanting, in Petrolina, Brazil.


The production of some compounds of the secondary metabolism in the bark of the L. ferrea stem was favored by mycorrhization with an increase in the concentration of flavonoids from 236% and 186%, respectively, in relation to the control, for the treatments with A. longula and C. etunicatum (Table 2). In plants inoculated with A. longula, the concentration of total tannins was increased in 47% in relation to the control, a benefit that did not occur in the treatments with G. albida and C. etunicatum (Table 2). The concentration of total phenols did not differ among the treatments; in contrast, the concentration of proanthocyanidins, in the plants associated with A. longula was lower than that recorded for the control (Table 2).


Table 2
Concentrations of total phenols, total flavonoids, total tannins and total proanthocyanidins in the stem bark of Libidibia ferrea, in the field, inoculated or non-inoculated with arbuscular mycorrhizal fungi (AMF), 13 months after transplanting, in Petrolina, Brazil.


4. DISCUSSION

Although the mycorrhizal colonization had indicated no differences between the treatments with inoculation and the control treatment (Table 1), it is possible to assume that the inoculated AMF were more efficient than the native AMF, considering that they favored the increase in the root diameter of mycorrhizal plants (Table 1). Similarly, a field study by Singh et al. [36Singh R, Kalra A, Ravish BS, Divya S, Parameswaran TN, Srinivas KV, et al. Effect of potential bioinoculants and organic manures on root-rot and wilt, growth, yield and quality of organically grown Coleus forskohlii in a semiarid tropical region of Bangalore (India). Plant Pathol 2012; 61: 700-8.
[http://dx.doi.org/10.1111/j.1365-3059.2011.02567.x]
] in semi-arid conditions recorded that the inoculation with Rhizophagus fasciculatus favored the development of Coleus forskohlii Briq. Other studies with legumes had also reported such benefit with the inoculated AMF favoring the increase of species and, in contrast with our study, producing more mycorrhizal structures than the control treatment [37Selvaraj T, Sumithra P. Effect of Glomus aggregatum and plant growth promoting rhizomicroorganisms on growth, nutrition and content of secondary metabolites in Glycyrrhiza glabra L. Indian J Appl Pure Biol 2011; 26: 283-90.]. However, in seedlings of Inga vera, legume occurring in the caatinga, mycorrhized with A. longula, G. albida, and C. etunicatum [38Lima CS, Campos MA, Silva FS. Mycorrhizal Fungi (AMF) increase the content of biomolecules in leaves of Inga vera willd. Symbiosis 2015; 65: 117-23.
[http://dx.doi.org/10.1007/s13199-015-0325-3]
] as well as in inoculated L. ferrea established in the field [27Silva FA, Ferreira MR, Soares LA, Sampaio EV, Silva FS, Maia LC. Arbuscular mycorrhizal fungi increase gallic acid production in leaves of field grown Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz. J Med Plants Res 2014; 8: 1110-5. b
[http://dx.doi.org/10.5897/JMPR2013.5503]
], the inoculation with AMF did not result in difference in the root diameter in relation to the non-inoculated control. This benefit may be associated with the presence of arbuscules in the inoculated roots since such structure favors the exchange of nutrients in the roots [39Peterson RL, Massicotte HB, Melville LH. Mycorrhizas : Anatomy and Cell Biology Canada 2004.], resulting in greater development.

In this study, the density of glomerospores in the plants mycorrhized with A. longula and C. etunicatum was higher than in the non-inoculated plants (Table 1). Similar results were recorded in inoculated C. forskohlii in field conditions with the density of the spores in the mycorrhized plants presenting differences in relation to the plants in the control treatment without inoculation and containing vermicompost [25Singh R, Soni SK, Kalra A. Synergy between Glomus fasciculatum and a beneficial Pseudomonas in reducing root diseases and improving yield and forskolin content in Coleus forskohlii Briq. Under organic field conditions. Mycorrhiza 2013; 23(1): 35-44.
[http://dx.doi.org/10.1007/s00572-012-0447-x] [PMID: 22648372]
]. Silva et al. [27Silva FA, Ferreira MR, Soares LA, Sampaio EV, Silva FS, Maia LC. Arbuscular mycorrhizal fungi increase gallic acid production in leaves of field grown Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz. J Med Plants Res 2014; 8: 1110-5. b
[http://dx.doi.org/10.5897/JMPR2013.5503]
] recorded results that differ from our study considering that the density of the spores in the rhizosphere of L. ferrea, in field conditions, did not differ among the inoculation treatments. The higher production of these propagules in the soil guarantees new colonization sites in the L. ferrea since favorable conditions may cause the spores to germinate and colonize the host [40Moreira FM, Siqueira JO. Micorrizas Microbiologia e bioquímica do solo Lavras: UFLA 2006.].

The study was pioneer at verifying an increase in the concentration of flavonoids and tannins in the bark of the stem due to mycorrhization, with emphasis to fungi A. longula (Table 2). Similar results had been reported regarding leaves of Pogostemon patchouli Pellet, inoculated with native fungi presenting increase in the concentration of flavonoids and tannins [11Selvaraj T, Nisha MC, Rajeshkumar S. Effect of indigenous arbuscular mycorrhizal fungi on some growth parameters and phytochemical constituents of Pogostemon patchouli Pellet. J Sci Technol 2009; 3: 222-34.]; in roots of Glycyrrhiza glabra L. a legume species, mycorrhization also increased the concentration of these compounds [37Selvaraj T, Sumithra P. Effect of Glomus aggregatum and plant growth promoting rhizomicroorganisms on growth, nutrition and content of secondary metabolites in Glycyrrhiza glabra L. Indian J Appl Pure Biol 2011; 26: 283-90.]; the shoots of mycorrhized Viola tricolor L. presented an increase in the concentration of flavonoid and rutin in relation to the non-inoculated control [41Zubek S, Rola K, Szewczyk A. Enhanced concentrations of elements and secondary metabolites in Viola tricolor L. induced by arbuscular mycorrhizal fungi. Plant Soil 2015; 390: 129-42.
[http://dx.doi.org/10.1007/s11104-015-2388-6]
]. Seedlings of native legume species of caatinga biome also presented an increase in the concentration and foliar content of flavonoids and tannins [38Lima CS, Campos MA, Silva FS. Mycorrhizal Fungi (AMF) increase the content of biomolecules in leaves of Inga vera willd. Symbiosis 2015; 65: 117-23.
[http://dx.doi.org/10.1007/s13199-015-0325-3]
, 42Pedone-Bonfim MV, Lins MA, Coelho IR, Santana AS, Silva FS, Maia LC. Mycorrhizal technology and phosphorus in the production of primary and secondary metabolites in cebil (Anadenanthera colubrina (Vell.) Brenan) seedlings. J Sci Food Agric 2013; 93(6): 1479-84.
[http://dx.doi.org/10.1002/jsfa.5919] [PMID: 23108717]
].

In contrast with the records in this study (Table 2), Silva et al. [5Silva FA, Silva FS, Maia LC. Biotechnical application of arbuscular mycorrhizal fungi used in the production of foliar biomolecules in ironwood seedlings. J Med Plants Res 2014; 8: 814-9. a [Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz var. ferrea].
[http://dx.doi.org/10.5897/JMPR2014.5358]
] verified in greenhouse that the foliar concentration of tannins indicated no differences among the treatments. In L. ferrea established in the field, the foliar concentration of tannins presented no difference caused by mycorrhization [27Silva FA, Ferreira MR, Soares LA, Sampaio EV, Silva FS, Maia LC. Arbuscular mycorrhizal fungi increase gallic acid production in leaves of field grown Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz. J Med Plants Res 2014; 8: 1110-5. b
[http://dx.doi.org/10.5897/JMPR2013.5503]
], such records are different from ours considering that the inoculation provided increase in the concentration of tannins in the bark of the stem of L. ferrea.

These results suggest that the production of compounds of the secondary metabolism in response to the inoculation with AMF may vary in a single species according to the age of the plants, the part studied, and the type of experimental condition. Furthermore, the increase in the concentration of these biomolecules can be caused by the increase in the concentration of precursors of these compounds, such as the gallic acid, which can be optimized through mycorrhizal inoculation in L. ferrea [27Silva FA, Ferreira MR, Soares LA, Sampaio EV, Silva FS, Maia LC. Arbuscular mycorrhizal fungi increase gallic acid production in leaves of field grown Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz. J Med Plants Res 2014; 8: 1110-5. b
[http://dx.doi.org/10.5897/JMPR2013.5503]
]; in addition, the activity of key-enzymes such as chalcone synthase, which benefits the formation of flavonoids, can be increased with a few inoculated treatments [20Zhang RQ, Zhu HH, Zhao HQ, Yao Q. Arbuscular mycorrhizal fungal inoculation increases phenolic synthesis in clover roots via hydrogen peroxide, salicylic acid and nitric oxide signaling pathways. J Plant Physiol 2013; 170(1): 74-9.
[http://dx.doi.org/10.1016/j.jplph.2012.08.022] [PMID: 23122788]
].

In accordance with the results by Silva et al. [27Silva FA, Ferreira MR, Soares LA, Sampaio EV, Silva FS, Maia LC. Arbuscular mycorrhizal fungi increase gallic acid production in leaves of field grown Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz. J Med Plants Res 2014; 8: 1110-5. b
[http://dx.doi.org/10.5897/JMPR2013.5503]
], who studied the concentration of phenols in leaves of L. ferrea, seven months after the transplanting, the concentration of phenols in the bark of the stem did not differ among the treatments (Table 2). In contrast, Riter Netto et al. [19Riter Netto AF, Freitas MS, Martins MA, Carvalho AJ, Vitorazi Filho JÁ. Efeito de fungos micorrízicos arbusculares na bioprodução de fenóis totais e no crescimento de Passiflora alata Curtis. Rev Bras Pl Med 2014; 16: 1-9.
[http://dx.doi.org/10.1590/S1516-05722014000100001]
] developed a study using screens and reported an increase in the foliar content of total phenols in Passiflora alata Curtis inoculated with C. etunicatum, Rhizophagus intraradices, and mixed inoculum (Rhizophagus clarus and Gigaspora margarita) in relation to the non-inoculated control. Other field studies that differ from ours indicate that inoculation with AMF provided an increase in these compounds, as recorded in flowers of Cynara cardunculus L. var. scolymus F. mycorrhized with Funneliformis. mosseae and R. intraradices [24Ceccarelli N, Curadi M, Martelloni L, Sbrana C, Picciarelli P, Giovannetti M. Mycorrhizal colonization impacts on phenolic content and antioxidant properties of artichoke leaves and flower heads two years after field transplant. Plant Soil 2010; 335: 311-23.
[http://dx.doi.org/10.1007/s11104-010-0417-z]
] and plants of Olea europaea L. inoculated with R. intraradices, with an increase in the concentration of total phenols in their fruits and alterations in the phenolic composition of the oil extracted [26Kara Z, Arslan D, Güler M, Güler S. Inoculation of arbuscular mycorrhizal fungi and application of micronized calcite to olive plant: Effects on some biochemical constituents of olive fruit and oil. Sci Hortic (Amsterdam) 2015; 185: 219-27.
[http://dx.doi.org/10.1016/j.scienta.2015.02.001]
]. It suggest that the benefit of mycorrhization in the production of metabolites may vary according to the plant species used, the part of the plant studied, and the AMF.

Other studies on legumes from the caatinga inoculated with AMF reported an increase in the foliar concentration of phenolic compounds [38Lima CS, Campos MA, Silva FS. Mycorrhizal Fungi (AMF) increase the content of biomolecules in leaves of Inga vera willd. Symbiosis 2015; 65: 117-23.
[http://dx.doi.org/10.1007/s13199-015-0325-3]
, 42Pedone-Bonfim MV, Lins MA, Coelho IR, Santana AS, Silva FS, Maia LC. Mycorrhizal technology and phosphorus in the production of primary and secondary metabolites in cebil (Anadenanthera colubrina (Vell.) Brenan) seedlings. J Sci Food Agric 2013; 93(6): 1479-84.
[http://dx.doi.org/10.1002/jsfa.5919] [PMID: 23108717]
]. Seedlings of cebil (Anadenanthera colubrina (Vell.) Brenan) had an increase in the concentration of phenols, flavonoids, and tannins due to the inoculation of mix (G. albida and A. longula) in relation to the non-inoculated control [42Pedone-Bonfim MV, Lins MA, Coelho IR, Santana AS, Silva FS, Maia LC. Mycorrhizal technology and phosphorus in the production of primary and secondary metabolites in cebil (Anadenanthera colubrina (Vell.) Brenan) seedlings. J Sci Food Agric 2013; 93(6): 1479-84.
[http://dx.doi.org/10.1002/jsfa.5919] [PMID: 23108717]
]. Oliveira et al. [8Oliveira PT, Alves GD, Silva FA, Silva FS. Foliar bioactive compounds in Amburana cearensis (Allemao) A. C. Smith seedlings : Increase of biosynthesis using mycorrhizal technology. J Med Plants Res 2015; 9: 712-8.
[http://dx.doi.org/10.5897/JMPR2015.5798]
] also reported that seedlings of inoculated Amburana cearensis (Allemao) A. C. Smith presented increase in the foliar concentration of tannins, flavonoids and phenols, with emphasis to fungi C. etunicatum. It is possible to infer that the application AMF results in different benefits to the production of groups of phenolic compounds, which depends on the fungi species employed, corroborating the initial hypothesis of our study.

This study was pioneer at recording the production of proanthocyanidins in plants inoculated with AMF. The lower concentration of these biomolecules in plants mycorrhized with A. longula (Table 2) is probably related to the accumulation of flavonoids in the plants of this treatment considering that some precursor molecules of the proanthocyanidins, with emphasis to the units flavan-3-ol, are also molecules involved in the biosynthesis of flavonoids [43Vermerris W, Nicholson R. Phenolic Compound Biochemistry 2006., 44Haslam E. Vegetable tannins - lessons of a phytochemical lifetime. Phytochemistry 2007; 68(22-24): 2713-21.
[http://dx.doi.org/10.1016/j.phytochem.2007.09.009] [PMID: 18037145]
]. Therefore, we may infer that these precursors can have been used by the plant metabolism in the formation of flavonoids resulting in the lower concentration of proanthocyanidins verified.

The accumulation of biomolecules of the secondary plant metabolism in response to symbiosis may be attributed to mechanisms as the improvement of the nutritional condition of the host [18Zimare SB, Borde MY, Jite PK, Malpathak NP. Effect of AM Fungi (Gf, Gm) on Biomass and Gymnemic Acid Content of Gymnema sylvestre (Retz.) R. Br. ex Sm. Proc Natl Acad Sci, India, Sect B Biol Sci 2013; 83: 439-45.
[http://dx.doi.org/10.1007/s40011-013-0159-9]
, 19Riter Netto AF, Freitas MS, Martins MA, Carvalho AJ, Vitorazi Filho JÁ. Efeito de fungos micorrízicos arbusculares na bioprodução de fenóis totais e no crescimento de Passiflora alata Curtis. Rev Bras Pl Med 2014; 16: 1-9.
[http://dx.doi.org/10.1590/S1516-05722014000100001]
], activation of metabolic routes [21Lohse S, Schliemann W, Ammer C, Kopka J, Strack D, Fester T. Organization and metabolism of plastids and mitochondria in arbuscular mycorrhizal roots of Medicago truncatula. Plant Physiol 2005; 139(1): 329-40.
[http://dx.doi.org/10.1104/pp.105.061457] [PMID: 16126866]
], production of signaling molecules [20Zhang RQ, Zhu HH, Zhao HQ, Yao Q. Arbuscular mycorrhizal fungal inoculation increases phenolic synthesis in clover roots via hydrogen peroxide, salicylic acid and nitric oxide signaling pathways. J Plant Physiol 2013; 170(1): 74-9.
[http://dx.doi.org/10.1016/j.jplph.2012.08.022] [PMID: 23122788]
], alterations in the activity of key-enzymes for the production of these compounds [20Zhang RQ, Zhu HH, Zhao HQ, Yao Q. Arbuscular mycorrhizal fungal inoculation increases phenolic synthesis in clover roots via hydrogen peroxide, salicylic acid and nitric oxide signaling pathways. J Plant Physiol 2013; 170(1): 74-9.
[http://dx.doi.org/10.1016/j.jplph.2012.08.022] [PMID: 23122788]
], hormonal alterations [45Mandal S, Upadhyay S, Wajid S, et al. Arbuscular mycorrhiza increase artemisinin accumulation in Artemisia annua by higher expression of key biosynthesis genes via enhanced jasmonic acid levels. Mycorrhiza 2015; 25(5): 345-57.
[http://dx.doi.org/10.1007/s00572-014-0614-3] [PMID: 25366131]
], increase in the expression of genes involved in the biosynthesis of these biomolecules [22Mandal S, Upadhyay S, Singh VP, Kapoor R. Enhanced production of steviol glycosides in mycorrhizal plants: a concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes. Plant Physiol Biochem 2015; 89: 100-6.
[http://dx.doi.org/10.1016/j.plaphy.2015.02.010] [PMID: 25734328]
, 45Mandal S, Upadhyay S, Wajid S, et al. Arbuscular mycorrhiza increase artemisinin accumulation in Artemisia annua by higher expression of key biosynthesis genes via enhanced jasmonic acid levels. Mycorrhiza 2015; 25(5): 345-57.
[http://dx.doi.org/10.1007/s00572-014-0614-3] [PMID: 25366131]
]. Such mechanisms have also been suggested in other studies [8Oliveira PT, Alves GD, Silva FA, Silva FS. Foliar bioactive compounds in Amburana cearensis (Allemao) A. C. Smith seedlings : Increase of biosynthesis using mycorrhizal technology. J Med Plants Res 2015; 9: 712-8.
[http://dx.doi.org/10.5897/JMPR2015.5798]
, 38Lima CS, Campos MA, Silva FS. Mycorrhizal Fungi (AMF) increase the content of biomolecules in leaves of Inga vera willd. Symbiosis 2015; 65: 117-23.
[http://dx.doi.org/10.1007/s13199-015-0325-3]
].

The results obtained from this study indicate that some of the above mentioned mechanisms have probably led to the increase in the production of flavonoids and tannins. The inoculation increase the concentration of precursors of these biomolecules, such as the Gallic acid, precursor of certain tannins [43Vermerris W, Nicholson R. Phenolic Compound Biochemistry 2006.], which in this plant species had an increase reported in the leaves [27Silva FA, Ferreira MR, Soares LA, Sampaio EV, Silva FS, Maia LC. Arbuscular mycorrhizal fungi increase gallic acid production in leaves of field grown Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz. J Med Plants Res 2014; 8: 1110-5. b
[http://dx.doi.org/10.5897/JMPR2013.5503]
] and the chalcones, precursors of flavonoids [43Vermerris W, Nicholson R. Phenolic Compound Biochemistry 2006.]. Furthermore, the symbiosis may have activated the expression of genes coding for enzymes related to the production of these compounds, such as the Phenylalanine-ammonia-lyase (PAL) and Chalcone synthase (Chs) associated with the production of flavonoids, which presented an increase in our study (Table 2) with a consequent improvement of the key-enzymes activity. Therefore, it is important to develop studies to quantify precursor molecules using, for example, High-efficiency Pressure Liquid Chromatography (HPLC), in order to analyze gene expression employing techniques such as real-time PCR. The use of biotechnological protocol considering the application of AMF may be an alternative to increase the production of biomolecules of the secondary metabolism in the bark of the stem of L. ferrea, which may add value to the phytomass to be commercialized by the industry of herbal medicines. Further studies on different plant parts, such as flowers, fruits are required.

CONCLUSION

Inoculation with A. longula may be an alternative to increase the production of biomolecules of the secondary metabolism in the bark of the L. ferrea stem in field conditions.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No Animals/Humans were used for studies that are base of this research.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENT

The authors would like to thank the FACEPE (Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico- Proc. n. 307749/2015-0) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for granting the main author with a scholarship.

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Endorsements



"Open access will revolutionize 21st century knowledge work and accelerate the diffusion of ideas and evidence that support just in time learning and the evolution of thinking in a number of disciplines."


Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


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