Benchmark Histological Studies of Mycorrhized Roots and Mycorrhizal-Like-Structures in Roots

Carla Ragonezi 1,* and Maria Amely Zavattieri 1,2,3 ID

1 Banco de Germoplasma ISOPlexis, Campus da Penteada, Universidade da Madeira, 9020-105 Funchal, Portugal; [email protected] 2 Departamento de Biologia, Pólo da Mitra Apartado 94, 7002-554 Évora, Portugal 3 Instituto de Ciências da Terra (ICT), Colégio Luís António Verney, Rua Romão Ramalho 59, 7000-671 Évora, Portugal * Correspondence: [email protected]; Tel.: +351-925193860 or +351-291705000 (ext. 5408)

 Received: 6 July 2018; Accepted: 29 August 2018; Published: 5 September 2018 

Abstract: Several studies have shown the potential of using Ectomycorrhizal (ECM) fungi in conifer micropropagation to overcome the cessation of adventitious root development. In vitro inoculation promotes the re-growth of the root system induced previously by auxin treatments, facilitating acclimation and diminishing the losses of plants because of a weak root system that is incapable of water and nutrient absorption. During a series of mycorrhization experiments, cryostat and ultrafine cuts were used to study the morpho-histological transformation of the symbiotic roots. To obtain cryostat cuts from pine roots a method frequently used for animal tissue was adopted. Molecular methods allowed fungi identification in all the mycorrhization phases and in the acclimation of derived plants. Mycorrhizal-like-structures derived from in vitro culture and axenic liquid cultures of roots were microscopically analyzed and compare with mycorrhizal roots.

Keywords: ectomycorrhiza; -like structures; ; adventitious roots; Hartig net

1. Introduction Ectomycorrhizal fungi (ECM fungi) are phylogenetically very diverse and more than 2000 species of ECM fungi worldwide have been identified, primarily from and Ascomycota. ECM fungi are associated mainly with woody perennials, including Pinaceae, Betulaceae, Fagaceae, and Diperocarpaceae in tropical, subtropical, and arid environments, and are regarded as key organisms in nutrient and carbon cycles in forest ecosystems [1]. Most fine roots and ECM fungi of trees are aggregated in the uppermost 20 cm of soil where nutrient circulation is high [2]. Ectomycorrhizas are characterized primarily by the presence of a sheath or a mantle of fungal tissue around the root, the Hartig-net, consisting of modified fungal hyphae that develops between root cells and a long system of fungal hyphae that connects the soil with the fruit bodies of the fungi forming the ectomycorrhizas [3,4]. This relates not only to the extent of root colonization but also to the development of hyphae in the soil. This mutualistic relationship with ECM fungi grants conifers an ecological advantage to withstand harsh living conditions. The way to recognize an ectomycorrhiza is through the structural modification of roots (dichotomous, coralloid or short monopodial roots) that are totally different from non-mycorrhized roots [5]. Another way to recognize them is via histological analysis in which the fungal mantle and Hartig-net are observed. Recently, molecular techniques display the possibilities to identify, by gene expression, the presence of . Antibodies and cDNA probes of the genes PF6.2 and LbRas of the mycorrhiza Laccaria bicolor, were also expressed in several other fungi that form mycorrhiza with red pine (Pinus resinosa)[6].

Methods and Protoc. 2018, 1, 34; doi:10.3390/mps1030034 www.mdpi.com/journal/mps Methods and Protoc. 2018, 1, 34 2 of 10

Biotechnological research for the clonal propagation of stone pine (Pinus pinea L.) was done previously in Portuguese research, since this important Mediterranean pine constitutes a relevant resource for the Portuguese economy mostly due to its edible seeds. After obtaining in vitro microshoots from mature cotyledons, it was possible to induce roots by the addition of growth regulators in the culture medium and modifying some physical parameters [7,8], but after the expression phase roots stop growing. This fact reduces the possibilities for acclimation of the micro pine plants and the clonal production within a commercial scale. To overcome this limitation a co-culture system with ectomycorrhizal fungi was developed [9]. The successful co-culture system methodology between stone pine plants with several ectomycorrhizal fungi belonging to the genus Lactarius, Pisolithus, Laccaria and Hebeloma, was patented under the Portuguese patent number PT 105239 [10]. To evaluate and validate the efficiency of the co-culture system, visual examination of the morphological changes of the root system was made during the acclimation phase in mixed substrate and in Rhizotrons, which allowed the direct observation of modified pine roots by different fungi. However, direct observation was sometimes not enough even for a trained researcher and thus microscopic analysis of the root was necessary. The morphological modifications of the root structure include monopodial, dichotomous, and/or coralloid branching of lateral roots; inhibition of root hair formation; and enlargement of cortical cells. During in vitro rooting, mycorrhizal-like structures, very similar to EMC colonized roots, appeared in some of the inoculated clones. These root modifications, very similar to mycorrhizas, appeared preferentially in jars left for a long time without any new media transference, and therefore in a situation of stress, possibly water stress due to culture media desiccation. These mycorrhiza-like structures were reported before in nature in [11], Pinus mugo [12], Pinus taeda, Pinus muricata, and [13]. Different pine species show different abilities to form mycorrhiza-like structures. Pinus pinea is not the exception. The objective of this paper is to focus on protocols and methods followed or adapted from other protocols to study the morpho-anatomical root changes derived from ectomycorrhizal inoculation and mycorrhizal-like-structures. Evaluation of the presence or absence of ectomycorrhizas in the modified roots of P. pinea was possible by using ultra-microtome cuts and cryostat micro cuts. The cryostat samples of the roots were obtain preparing the tissue according to a methodology generally used for preparation of animal tissue samples as described in material and methods.

2. Material and Methods

2.1. Plant Material Mature seeds of stone pine were obtained from selected plus trees (Alcácer do Sal, Portugal) and were stored in a cold chamber at 4 ◦C until used. For the details of in vitro shoot induction from cotyledons of mature seeds and shoot multiplication see [14]. Microshoots were placed in rooting medium WPMRI (Wood Plant Medium root induction) and after, in WPMRE (Wood Plant Medium root expression) [15].

2.2. Fungi Sporocarps of several ectomycorrhizal fungi including Pisolithus arhizus (Scop.) Rauschert, Hebeloma cylindrosporum Romagn., Russula torulosa Bres., Lactarius deliciosus (L.) Gray., Rhizopogon luteolus Fr., Laccaria laccata (Scop.) Cooke, bellinii (Inzenga) Kuntze were collected from a pure stand of stone pine in Portugal (N 38◦250; W 7◦560) in winter after a few days of heavy rainfall. In situ was done the preliminary identification via morphological traits and specimens were stored at 4 ◦C prior to sterilization and isolation procedures. Methods and Protoc. 2018, 1, 34 3 of 10

2.3. Mycelia Isolation and Fungal Cultures The methodology used was according to [16], briefly, the fruiting bodies (sporocarps) were cut into pieces and disinfected by placed in running water for 10 min and then in 70% ethanol for 2 min. Then, pieces were rinsed with sterile distilled water in a laminar flow chamber, placed in 20% (v/v) commercial bleach (≤5% active chlorine) for 10 min and rinsed four times with sterile water. Larger Methods Protoc. 2018, 1, x FOR PEER REVIEW 3 of 10 pieces were then cut in smaller pieces (50 mm3) for growth. The isolation and culture of the ECM fungi speciespieces was were made then in cut modified in smaller Hagen pieces medium (50 mm3 [)17 for]. growth. The formulation The isolation of and modified culture Hagen of the ECM (per liter of medium)fungi species was: was KH 2madePO4 0.5in modified g, NH4CL Hagen 0.5 g,medium MgSO [17].4·7H The2O 0.5formulation g, FeCL 3of(1%) modified 0.5 mL, Hagen glucose (per 5 g, malt extractliter of medium) 5 g, thiamine was: KH HCL2PO 504 0.5µg, g, and NH4 agarCL 0.5 15 g, g. MgSO The pH4·7H was2O 0.5 adjusted g, FeCL3to (1%) 4.5–5.0. 0.5 mL, The glucose media 5 was autoclavedg, malt for extract 20 min 5 g, atthiamine 121 ◦C, HCL 1 atm, 50 andμg, and 100 agar mg mL15 g.−1 Theof RifampicinpH was adjusted (Sigma-Aldrich to 4.5–5.0. The®, Saint media Louis, was autoclaved for 20 min at 121 °C, 1 atm, and 100 mg mL−1 of Rifampicin (Sigma-Aldrich®, Saint MO, USA) was added to the media after cooling to avoid the contamination by bacteria. Pieces Louis, MO, USA) was added to the media after cooling to avoid the contamination by bacteria. Pieces of sporocarps in this medium were kept at 25 ◦C in the dark and subculture at weekly intervals. of sporocarps in this medium were kept at 25 °C in the dark and subculture at weekly intervals. After After14 14 days days in in culture culture the the mycelia mycelia were were used used for co-cultures for co-cultures (Figure (Figure 1a). 1a).

Figure 1. In vitro co-culture system between stone pine microshoots with some developed roots Figure 1. In vitro co-culture system between stone pine microshoots with some developed roots induced by 2-Naphthaleneacetic acid (NAA) and Pisolithus sp. (white arrow) in the surface of the induced by 2-Naphthaleneacetic acid (NAA) and Pisolithus sp. (white arrow) in the surface of the double layer medium (a). Acclimation of mycorrhized pine plants in vermiculite (b) and in mixed doublesubstrates layer medium (c) from (whicha). Acclimation root samples of were mycorrhized taken for cryostat pine plants analysis. in vermiculite Some mycorrhized (b) and plants in mixed substrateswere (transferredc) from which to rhizotrons root samples for direct were observ takenation for cryostatof mycorrhizal analysis. morphological Some mycorrhized structures plants (d). were transferred to rhizotrons for direct observation of mycorrhizal morphological structures (d). 2.4. In Vitro Co-Culture 2.4. In VitroFollowing Co-Culture the in vitro rooting induction and expression phase, pine roots generally stop the Followinggrowth, this the is anin evidence vitro rooting of recalcitrance induction in and this expressionspecies and the phase, main pine reason roots to use generally mycorrhizal stop the inoculation. In a study by Oliveira et al. [14], it was demonstrated that the co-culture system was growth, this is an evidence of recalcitrance in this species and the main reason to use mycorrhizal extremely useful to cope with the difficulties of rooting. Microshoots were transferred to the double- inoculation. In a study by Oliveira et al. [14], it was demonstrated that the co-culture system layer medium and, after a brief period of adaptation into the medium, inoculated with selected fungi. was extremelyControl plants useful were to also cope transferred with the to difficulties a double-layer of rooting.medium but Microshoots were not inoculated. were transferred to the double-layer medium and, after a brief period of adaptation into the medium, inoculated with selected fungi.2.5. Control Acclimation plants were also transferred to a double-layer medium but were not inoculated. After the co-culture, plants of all treatments including control plants (without mycorrhization) 2.5. Acclimation went through acclimation with the aim to follow the ex vitro growth of the root system. The plantlets Afterwere first the co-culture,transferred to plants sterile of vermiculite all treatments for tw includingo weeks and control then transferred plants (without to a mixed mycorrhization) substrate wentof through vermiculite/perlite/peat acclimation with in the a proportion aim to follow of 2:1:1. the ex The vitro acclimation growth oflasted the root10 weeks system. in a The growth plantlets −2 −1 werechamber first transferred at 25/19 °C to day/night sterile vermiculite temperatures, for with two 16 weeks h photoperiod and then (270 transferred μmol m tos ) a and mixed a relative substrate humidity of 80%. Glass jars were covered with plastic film in which some holes were made to ensure of vermiculite/perlite/peat in a proportion of 2:1:1. The acclimation lasted 10 weeks in a growth gas exchange and to ensure a rapid acclimation. A high 80% relative humidity was maintained in the chamber at 25/19 ◦C day/night temperatures, with 16 h photoperiod (270 µmol m−2 s−1) and a relative growth chamber, during the first week and was gradually decreased to 60%. Plants were watered as humidityrequired, of 80%. alternating Glass jars between were sterile covered water with and plastic liquid filmWPM in (macronutrients which some holes only). were made to ensure gas exchange and to ensure a rapid acclimation. A high 80% relative humidity was maintained in the growth2.6. chamber, Rhizotron during the first week and was gradually decreased to 60%. Plants were watered as required, alternatingIn most of the between experiment, sterile some water clonal and pl liquidants inoculated WPM (macronutrients with ectomycorrhizal only). fungi Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) SF Gray, were transferred to rhizotrons. Rhizotrons allow the visualization of root development and changes in the appearance of the root derived from different fungi colonization, whenever desired without disturbing the normal functions of the plants. Basically, they are made of two acrylic plates, 20 × 20 cm each, with intervals made by 5 mm spacers and filled with Sphagnum sterilized turf, to support and feed the plants [18], with some adaptations later proposed [19]. Observation of the roots was performed from 4 to 6 weeks, which is the period required for most plants to explore the available space.

Methods and Protoc. 2018, 1, 34 4 of 10

2.6. Rhizotron In most of the experiment, some clonal plants inoculated with ectomycorrhizal fungi Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) SF Gray, were transferred to rhizotrons. Rhizotrons allow the visualization of root development and changes in the appearance of the root derived from different fungi colonization, whenever desired without disturbing the normal functions of the plants. Basically, they are made of two acrylic plates, 20 × 20 cm each, with intervals made by 5 mm spacers and filled with Sphagnum sterilized turf, to support and feed the plants [18], with some adaptations later proposed [19]. Observation of the roots was performed from 4 to 6 weeks, which is the period required for most plants to explore the available space.

2.7. Cryostat Mycorrhized root samples from the acclimation to the mixed substrates were collected at the end of each experiment and identified for histological and anatomical studies. Roots were fixed in 4% glutaraldehyde, diluted in 0.1 M HEPES buffer 2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid, pH 6.8 and stored in a refrigerator (at 4◦) for 24 h. Afterwards, roots were washed twice in 1N PBS phosphate buffer saline (PBS) with 4% sucrose for 15 min and finally washed in PBS with 15% sucrose. After washing, roots were placed in PBS with 15% sucrose and 7.5% of microbiology gelatin (Merck®, Lisbon, Portugal) for 1 to 2 h at 37 ◦C. Other Petri dishes with the same gelatin base were prepared and, after 1 h when the solution solidified, roots were placed on the surface of the gelatin and covered with a 1 cm layer of molten gelatin solution. Petri dishes were stored in a refrigerator for 1 h; blocks (1 cm × 1 cm × 1 cm) of gelatin with roots were cut out, frozen, and stored at −80 ◦C. Longitudinal and transverse sections, approximately 5–10 µm thick, were cut at −33 ◦C using a cryostat Leica CM3050 S (Leica Biosystems, Wetzlar, Germany), transferred to glass slides and stained with a common fountain pen ink. Sections were observed under an Olympus (Southend-on-Sea, UK) microscope at a magnification of 1125×.

2.8. Solution Gelatin For a 100 mL solution, gradually mix 7.5 g of microbiology gelatin (Merck®) powder with PBS (can be heated up to 55 ◦C) to go into solution. Add 15 g of sucrose and mix until dissolved (you can warm up to 55 ◦C to help). Set the volume with PBS and place at 37 ◦C in a hot air oven. Equilibrate the temperature for 30 min before placing the tissue in the gelatin.

2.9. Axenic Root Cultures and Microscopic Analysis To induce the formation of mycorrhizas-like-structures for comparison with symbiotic ectomycorrhizal structures the methodology used was according to [20], briefly, root segments of 2 cm long, obtained from germinated pine seeds (pinions), were excised and cultured in liquid medium in an orbital shaker (125 rpm) for 3 to 4 weeks. Afterward, the roots were photographed and used for histological studies. Two different methods were used (1) hand sections of root segments were obtained, segments were placed between pieces of laboratory Parafilm and cut as thin as possible using a razor blade [16] and observed under a Zoom Stereo Research Microscope 7-70X Olympus SZH10. Structural details of the root anatomy were observed under a Light Microscope with image acquisition device Olympus CX-40 and photographs were taken with a Canon (Tokio, Japan) Power-Shot A630 camera. (2) Dichotomous and short roots were fixed in FAA (dehydrated in an aqueous series of ethanol (70, 80, 95, 100%), clarified in xylol, embedded in paraffin, and cut with a rotary microtome (8–10 µm). The sections were stained with toluidine blue, mounted in Entellan® (rapid mountain medium for microscopy) and observed under a Light Microscope [21]. These methods were also used in mycorrhized roots as a complement of the previous described using a cryostat cuts and stain. Methods and Protoc. 2018, 1, 34 5 of 10

2.10. Identification of the Fungal Isolates The identification of the fungi was first made at the site of the collection of the sporocarps in the stands of pine. This characterization was based on their morphotypes. To confirm the identity of the collected material, internal transcribed spacer (ITS) amplification of the ribosomal genes was applied using the pure cultures derived from the sporocarps. Small subunit 18S, and the 5.8S of the rDNA repeat unit was amplified using the oligonucleotides primers ITS 5 and ITS4. Polymerase chain reaction (PCR) products were analyzed by gel electrophoresis and sequenced by capillary electrophoresis. The sequences of the ITS region were aligned with related fungal strains from the GenBank databases for the homology analysis. Also, M13-PCR fingerprinting methodology for monitoring different species of Basidiomycota and Ascomycota associated with stone pine was tested. For details about these methods used see Ragonezi et al. [22].

3. Results Methods Protoc. 2018, 1, x FOR PEER REVIEW 5 of 10 All the ECM fungi tested enhance rooting as it was expected, but the number of new roots, and 3. Results the length of them varied between ECM fungi tested. Higher number of roots and branches obtained by the presenceAll the ECM of the fungi mycorrhizal tested enhance fungi rooting was possible as it wa becauses expected, ECM but fungithe number exude of (among new roots, many and other chemicalthe length compounds) of them varied auxins, between that directly ECM fungi influence tested. theHigher root number growth. of roots Re-growth and branches and the obtained production of newby rootsthe presence and branches of the mycorrhiz is highlyal fungi advantageous was possible during because acclimation ECM fungi phaseexude (among of pine many plants. other Among chemical compounds) auxins, that directly influence the root growth. Re-growth and the production the ECM tested, Pisolithus arhizus was selected here to exemplify our results. Later it was also used of new roots and branches is highly advantageous during acclimation phase of pine plants. Among in thethe experiments ECM tested, Pisolithus on biochemical arhizus was signaling selected with here to microplants exemplify our of results. stone pineLater [it9 ].was The also co-culture used in of P. pineathe microshootsexperiments on with biochemicalPisolithus signalingsp. (Figure with microplants1a) effectively of stone helped pine [9]. to overcomeThe co-culture one ofof P. thepinea most commonmicroshoots problems with associated Pisolithus withsp. (Figurein vitro 1a)rooting: effectively the helped inhibition to overcome of root elongationone of the most under common the culture conditionsproblems and associated the adaptation with in of vitro the plantletsrooting: the to exinhibition vitro conditions. of root elongation Even when under an extremelythe culture poor substrateconditions like vermiculiteand the adaptation was used, of the during plantlets the to earlyex vitro phase conditions. of acclimation Even when (Figure an extremely1b) none poor of the inoculatedsubstrate plantlets like vermiculite died and was a vastused, mycorrhizal during the early symbiosis phase of establishment acclimation (Figure was observed1b) none of in the mixed substratesinoculated (Figure plantlets1c) and died in and rhizotrons a vast mycorrhizal (Figures1d symbiosis and2). Moreover, establishment fewer was roots observed were in lost mixed during transplantationsubstrates (Figure to mixed 1c) and substrates in rhizotrons which (Figures was facilitated 1d and 2). by Moreover, the morphological fewer roots were modifications lost during of the transplantation to mixed substrates which was facilitated by the morphological modifications of the mycorrhized roots such as the presence of the hyphae around the roots (mantle) (Figures2c and3) and mycorrhized roots such as the presence of the hyphae around the roots (mantle) (Figures 2c and 3) the internaland the Hartig-netinternal Hartig-net (Figure (Figure3a), which 3a), increasedwhich increased root thicknessroot thickness and contributedand contributed to ato more a more robust root systemrobust root [16 ].system [16].

a b c

Figure 2. Pine plant in the rhizotron (a) where it is easy to observe mycorrhizas and hyphae in the Figure 2. Pine plant in the rhizotron (a) where it is easy to observe mycorrhizas and hyphae in the turf substrate. Turf maintains an elevated humidity that favors the growth of fungi. Magnification of turf substrate. Turf maintains an elevated humidity that favors the growth of fungi. Magnification of mycorrhizas in a stereomicroscopy, 10×, scale bar 2 mm (b). Detail of dichotomous branching and × mycorrhizashairy white in hyphae a stereomicroscopy, 50x, scale bar 1 10mm ,(c scale). bar 2 mm (b). Detail of dichotomous branching and hairy white hyphae 50x, scale bar 1 mm (c). Samples of dichotomous and coralloid roots from the plants grown in mixed substrate and in Samplesrhizotrons of were dichotomous collected after and 10 coralloid weeks in acclim rootsation from for the a plantsdetailed grown histological in mixed study. substrate According and in rhizotronsto our wereprevious collected experience, after the 10 symbiotic weeks in structures acclimation were for highly a detailed variable histological in their complexity, study. According but the to transverse sections of the ectomycorrhizal roots showed a well-developed mantle and Hartig-net for our previous experience, the symbiotic structures were highly variable in their complexity, but the almost all ectomycorrhizal fungi tested (Figures 3a,b, 4 and 5). transverse sections of the ectomycorrhizal roots showed a well-developed mantle and Hartig-net for almost all ectomycorrhizal fungi tested (Figure3a,b, Figures4 and5).

Methods Protoc. 2018, 1, x FOR PEER REVIEW 5 of 10

3. Results All the ECM fungi tested enhance rooting as it was expected, but the number of new roots, and the length of them varied between ECM fungi tested. Higher number of roots and branches obtained by the presence of the mycorrhizal fungi was possible because ECM fungi exude (among many other chemical compounds) auxins, that directly influence the root growth. Re-growth and the production of new roots and branches is highly advantageous during acclimation phase of pine plants. Among the ECM tested, Pisolithus arhizus was selected here to exemplify our results. Later it was also used in the experiments on biochemical signaling with microplants of stone pine [9]. The co-culture of P. pinea microshoots with Pisolithus sp. (Figure 1a) effectively helped to overcome one of the most common problems associated with in vitro rooting: the inhibition of root elongation under the culture conditions and the adaptation of the plantlets to ex vitro conditions. Even when an extremely poor substrate like vermiculite was used, during the early phase of acclimation (Figure 1b) none of the inoculated plantlets died and a vast mycorrhizal symbiosis establishment was observed in mixed substrates (Figure 1c) and in rhizotrons (Figures 1d and 2). Moreover, fewer roots were lost during transplantation to mixed substrates which was facilitated by the morphological modifications of the mycorrhized roots such as the presence of the hyphae around the roots (mantle) (Figures 2c and 3) and the internal Hartig-net (Figure 3a), which increased root thickness and contributed to a more robust root system [16].

a b c

Figure 2. Pine plant in the rhizotron (a) where it is easy to observe mycorrhizas and hyphae in the turf substrate. Turf maintains an elevated humidity that favors the growth of fungi. Magnification of mycorrhizas in a stereomicroscopy, 10×, scale bar 2 mm (b). Detail of dichotomous branching and hairy white hyphae 50x, scale bar 1 mm (c).

Samples of dichotomous and coralloid roots from the plants grown in mixed substrate and in rhizotrons were collected after 10 weeks in acclimation for a detailed histological study. According to our previous experience, the symbiotic structures were highly variable in their complexity, but the Methodstransverse and Protoc. sections2018, 1, 34 of the ectomycorrhizal roots showed a well-developed mantle and Hartig-net for6 of 10 almost all ectomycorrhizal fungi tested (Figures 3a,b, 4 and 5).

Methods Protoc. 2018, 1, x FOR PEER REVIEW 6 of 10 Figure 3. Cryostat transversal section of pine root colonized by Pisolithus sp. showing the mantle Methods Protoc. 2018, 1, x FOR PEER REVIEW × µ 6 of 10 hyphaeFigure around 3. Cryostat the root transversal surface (M) section (200 of ),pine scale root bar colonized 20 m ( aby). InternalPisolithus Hartig-netsp. showing (HN). the mantle Details of the transverse section showing well-differentiated Hartig-net (HN) in cortical cells of the root (2000×). Figurehyphae 3. around Cryostat the transversal root surface section (M) (200×), of pine scale root bar colonized 20 μm(a ).by Internal Pisolithus Hartig-net sp. showing (HN). the Details mantle of µ b Scalehyphaethe bar transverse 7.5 aroundm ( section ).the root showing surface well-differentiated (M) (200×), scale bar Hart 20ig-net μm( a(HN)). Internal in cortical Hartig-net cells of (HN). the root Details (2000×). of theScale transverse bar 7.5 μ sectionm (b). showing well-differentiated Hartig-net (HN) in cortical cells of the root (2000×). EctomycorrhizalScale bar 7.5 μm structures(b). produced between pine plants and fungi during the acclimation Ectomycorrhizal structures produced between pine plants and fungi during the acclimation phase (derived from previous co-cultures) were analyzed by cryostat and ultramicrotome cuts. phase (derived from previous co-cultures) were analyzed by cryostat and ultramicrotome cuts. The The ectomycorrhizasEctomycorrhizal were structures morphologically produced very between different pine from plants each and other fungi in during the dichotomous the acclimation structure phaseectomycorrhizas (derived from were previous morphologically co-cultures) very were different analyzed from by each cryostat other and in the ultramicrotome dichotomous cuts.structure The color, hyphal extension and color of the mantle, and in the internal development of the Hartig net. ectomycorrhizascolor, hyphal extension were morphologically and color of the verymantle, different and in from the internal each other development in the dichotomous of the Hartig structure net. In In Figures4 and5 it is possible to observe two examples. color,Figures hyphal 4 and extension 5 it is possible and color to observe of the mantle,two examples. and in the internal development of the Hartig net. In Figures 4 and 5 it is possible to observe two examples.

10µm

Figure 4. Morphology of the ectomycorrhiza produced between the pine clone plant Fp2 inoculated with 10µm Figure 4. Morphology of the ectomycorrhiza produced between the pine clone plant Fp2 inoculated the 1N(1)b, scale bar 1 mm (a); longitudinal cryostat Section 5 mm (400×), with mantle withFigure the fungus 4. Morphology 1N(1)b, scaleof the barectomycorrhiza 1 mm (a); produced longitudinal between cryostat the pine Section clone plant5 mm Fp2 (400 inoculated×), with with mantle thedevelopment fungus 1N(1)b, scale bar scale 20 μbarm (b1); mmdetail ( ofa); thelongitudinal mantle hyphae, cryostat 1000×, Section scale bar5 mm 10 μ(400×),m(c); Hartig with netmantle and development scale bar 20 µm (b); detail of the mantle hyphae, 1000×, scale bar 10 µm (c); Hartig net developmentfungi between scale cell walls,bar 20 1000×, μm (b scale); detail bar of 10 the μ mmantle (d) mantle hyphae, and 1000×, Hartig scale net inbar transverse 10 μm(c); section Hartig obtained net and and fungi between cell walls, 1000×, scale bar 10 µm (d) mantle and Hartig net in transverse section fungiwith anbetween ultra-microtome cell walls, 1000×,(1 μm) scalestained bar with 10 μ Toluidinem (d) mantle blue, and scale Hartig bar 10 net μ inm transverse(e). section obtained µ µ obtainedwith an with ultra-microtome an ultra-microtome (1 μm) stained (1 m) with stained Toluidine with blue, Toluidine scale bar blue, 10 μm scale (e). bar 10 m (e).

Figure 5. Enlarge images of the ectomycorrhiza produced between pine clone plant Fo2 inoculated Figurewith fungus 5. Enlarge 1M (2)a, images scale of bar the 1 ectomycorrhiza mm (a); transverse produc sec ed(5 mm)between cryostat pine cutclone at 400×plant magnification Fo2 inoculated in Figure 5. Enlarge images of the ectomycorrhiza produced between pine clone plant Fo2 inoculated withmicroscope, fungus 1Mscale (2)a, bar scale 20 μ barm ( b1); mm mantle (a); transversehyphae 1000×, sec (5 scale mm) bar cryostat 10 μm cut (c) at Hartig 400× magnificationnet detail 1000×, in with fungus 1M (2)a, scale bar 1 mm (a); transverse sec (5 mm) cryostat cut at 400× magnification in microscope,scale bar 10 μscalem (d bar) mantle 20 μm and (b );Hartig-net mantle hyphae observed 1000×, in tran scalesverse bar 10sections μm (c )using Hartig an netultra-microtome detail 1000×, microscope,scale(1), scale bar scale 10bar μ 10m bar μ(dm) 20 mantle(e).µ m (andb); mantleHartig-net hyphae observed 1000 in× tran, scalesverse bar sections 10 µm using (c); Hartig an ultra-microtome net detail 1000 ×, scale(1), bar scale 10 µ mbar ( d10) mantleμm (e). and Hartig-net observed in transverse sections using an ultra-microtome (1), scaleExtensive bar 10 µm dichotomous (e). and coralloid branching of lateral roots occurred during in vitro rooting at theExtensive expression dichotomous phase in andcontrol coralloid plants branching (Figure 6a).of lateral Somehow, roots occurredthe question during of inwhether vitro rooting some mycelium could have contaminated the control plants was raised, even thought this was highly Extensiveat the expression dichotomous phase in and control coralloid plants branching (Figure 6a). of lateralSomehow, roots the occurred question during of whetherin vitro somerooting improbable. Also, non-inoculated plants that remained in the culture medium for longer than a at themycelium expression could phase have incontaminated control plants the control (Figure pl6antsa). was Somehow, raised, even the questionthought this of whetherwas highly some improbable.month, in a drierAlso, medium, non-inoculated developed plants numerous that rema mycorrhizal-likeined in the culture structures medium (Figure for 6b). longer This thanwould a mycelium could have contaminated the control plants was raised, even thought this was highly month,suggest in a a correlationdrier medium, between developed osmotic numerous and/or mycorrhizal-like nutritional stress structures and the (Figure abundance 6b). This of would these improbable.suggestmimicking a Also, correlationstructures. non-inoculated Thebetween hand-made osmotic plants sections thatand/or remainedobse nurvationtritional in confirmed thestress culture and the the mediumabsence abundance of for a mantle longerof these and than a month,mimickingHartig-net in a drier structures.in medium,this ectomycorrhizal-like-structur The developed hand-made numerous sections obse mycorrhizal-likees (Figurervation confirmed6c) and structures other the absenceimportant (Figure of a6internal mantleb). This andand would suggestHartig-netexternal a correlation microscopic in this ectomycorrhizal-like-structur between differences osmotic was found and/or compares nutritional(Figureing this 6c)structure stressand other with and importantdichotomous the abundance internal branching and of these mimickingexternalroots derived structures. microscopic from ectomycorrhizas. The differences hand-made was found sections compar observationing this structure confirmed with the dichotomous absence of branching a mantle and roots derived from ectomycorrhizas.

Methods and Protoc. 2018, 1, 34 7 of 10

Hartig-net in this ectomycorrhizal-like-structures (Figure6c) and other important internal and external microscopic differences was found comparing this structure with dichotomous branching roots derived fromMethods ectomycorrhizas. Protoc. 2018, 1, x FOR PEER REVIEW 7 of 10

Methods Protoc. 2018, 1, x FOR PEER REVIEW 7 of 10

a c b Figure 6. In vitro dichotomous branching of roots (arrows) with no fungi culture in the upper layer Figure 6. In vitro dichotomousa branching of roots (arrows) withb no fungi culture in the upper layerc medium (a). Dichotomous and coralloid branching (arrows) in plants that remain for a long-time in medium (a). Dichotomous and coralloid branching (arrows) in plants that remain for a long-time vitroFigure rooting 6. In vitro expression dichotomous medium branching (b) and of histological roots (arro ws)preparation with no fungiof a dichotomous culture in the root upper without layer in vitro rooting expression medium (b) and histological preparation of a dichotomous root without mantlemedium or ( aHartig-net,). Dichotomous scale barand 50coralloid μm (c). branching (arrows) in plants that remain for a long-time in mantle or Hartig-net, scale bar 50 µm (c). vitro rooting expression medium (b) and histological preparation of a dichotomous root without Themantle results or Hartig-net, in axenic scale liquid bar cultures50 μm (c ).showed that all pine clones tested can produce mycorrhiza- Thelike-structures results in(Figure axenic 7a,d). liquid Supplementary, cultures showed to exclude that any all possibility pine clones of the tested liquid cancultures produce mycorrhiza-like-structurescontaminationThe results with in axenic fungal (Figureliquid mycelium, cultures7a,d). several showed Supplementary, histological that all pine studies to clones exclude were tested carried any can possibility produce out, like mycorrhiza- hand-made of the liquid cuts between parafilm foil (Figure 7b) and ultra-microtomy cuts and subsequent staining as shown cultureslike-structures contamination (Figurewith 7a,d). fungal Supplementary, mycelium, to exclude several any histological possibility studiesof the liquid were cultures carried out, incontamination Figure 7c,f. with fungal mycelium, several histological studies were carried out, like hand-made like hand-made cuts between parafilm foil (Figure7b) and ultra-microtomy cuts and subsequent cuts between parafilm foil (Figure 7b) and ultra-microtomy cuts and subsequent staining as shown staining as shown in Figure7c,f. in Figure 7c,f.

Figure 7. Dichotomous branching in liquid axenic culture clones 122 (a) and clone 138 (d). Hand-made root cut using parafilm foil; the dichotomous structure did not have a mantle or a Hartig-net, scale barFigure 40 μ 7.m Dichotomous (b). Dichotomous branching roots in derived liquid axenicfrom axenic culture culture, clones without122 (a) and ma clonentle, or 138 hyphae (d). Hand-made around it, Figure 7. Dichotomous branching in liquid axenic culture clones 122 (a) and clone 138 (d). Hand-made scaleroot cutbar using 2 mm parafilm (e). Ultramicrotome foil; the dichotomous cuts of dichotomous structure did and not coralloid have a mantleroots stained or a Hartig-net, with toluidine scale root cutblue,bar 40 using no μ mmantle, ( parafilmb). Dichotomous nor Hartig-net foil; the roots dichotomous was derived observed, from structurescale axenic bar culture, 40 did μm not and without have scale maabar mantlentle, 30 μ orm orhyphae(c and a Hartig-net, f around). it, scale bar 40scaleµm bar (b). 2 Dichotomousmm (e). Ultramicrotome roots derived cuts of from dichotomous axenic culture, and coralloid without roots mantle, stained or with hyphae toluidine around it, scaleSpeciesblue, bar Identification 2no mm mantle, (e). nor Ultramicrotomeof P. Hartig-net arhizus and was L. cuts observed, deliciosus of dichotomous scale bar 40 μ andm and coralloid scale bar roots 30 μm stained (c and f with). toluidine blue,PCR no mantle, products nor of Hartig-net ITS4/ITS5 was primers, observed, were scale644 bp bar and 40 µ400m andbp obtained scale bar from 30 µm dikariontic (c and f). isolates fromSpecies Pisolithus Identification sp. P1001of P. arhizus and Lactarius and L. deliciosus deliciosus UEZB1, respectively. Sequence alignments of P. arhizusPCR showed products identities of ITS4/ITS5 that ranged primers, from were 99–100% 644 bp amongand 400 isolates bp obtained belonging from todikariontic P. arhizus isolates; for L. deliciosusfrom Pisolithus the homology sp. P1001 was and over Lactarius 99%. Both deliciosus sequences UEZB1, were respectively. published inSequence GenBank alignments with accession of P. numberarhizus showed HQ896485 identities and thatJQ066791, ranged respectivelyfrom 99–100% [2 3,24].among Results isolates demonstratedbelonging to P.that arhizus M13-PCR; for L. deliciosus the homology was over 99%. Both sequences were published in GenBank with accession number HQ896485 and JQ066791, respectively [23,24]. Results demonstrated that M13-PCR

Methods and Protoc. 2018, 1, 34 8 of 10

Species Identification of P. arhizus and L. deliciosus PCR products of ITS4/ITS5 primers, were 644 bp and 400 bp obtained from dikariontic isolates from Pisolithus sp. P1001 and Lactarius deliciosus UEZB1, respectively. Sequence alignments of P. arhizus showed identities that ranged from 99–100% among isolates belonging to P. arhizus; for L. deliciosus the homology was over 99%. Both sequences were published in GenBank with accession number HQ896485 and JQ066791, respectively [23,24]. Results demonstrated that M13-PCR discriminated between species and taxonomic groups. Based on the specific PCR fingerprints and the high interspecies variation of the banding patterns, a clear distinction among all species used for the test was viable. M13-PCR highlighted differentiation at the species and strain level [25].

4. Discussion Apparently, there was a strong morphological similarity between extensive dichotomous and coralloid branching of lateral roots that grew because of changes in the osmotic potential or nutrient content of the culture medium and those derived from fungal inoculation of pine plants. Due to this macroscopic similarity, it may be difficult to diagnose ectomycorrhizas without confirmation either by molecular analysis or by the ECM fungi status via histological analysis. The combination of sequence analysis of the ITS regions of the rDNA and the PCR fingerprinting technique was extremely helpful to identify the species collected in the field as well as to monitor the fungus involved in all the steps in our mycorrhization programs. M13-PCR is a rapid method for DNA amplification of polymorphic sequences. It was possible with M13 to characterize the genetic profile of sporocarps collected such as L. deliciosus, P. arhizus, and R. roseolus. The advantages of this DNA amplification method are simplicity, high levels of resolution, universal availability of the primers, reproducibility, easy database analysis, and the reduced costs [26]. PCR fingerprinting is very helpful to resolve taxonomic problems and to differentiate species and strains of filamentous fungi [27]. The presence of mycorrhiza-like-structures has been previously observed in other pine species and might be indicative of long coevolution of these two kingdoms for millions of years [13,28]. The presence of these structures was reported in other in vitro culture systems between ECM and conifers, for example, Picea abies cell culture induced ectomycorrhizal fungi to form mycorrhiza-like structures which normally are only generated in the presence of host roots [29]. It is unclear what is the natural compound associated with the change in root architecture, like that which occurs with strigolactones in the interaction promotion with arbuscular mycorrhizal fungi [30]. Histological observations allowed us to definitively separate structures derived from the symbiosis by the fungi of other similar structures induced by other factors (genetics; environmental). The adaptation of a protocol used for animal samples preparation showed to be simple and highly effective to be used for cryostat cuts. In this paper cuts made with the Pisolithus arhizus were shown. However, all the ECM fungi tested improved rooting of stone pine and microscopic observations confirmed, for all of them, a good development of the mantle and Hartig-net.

5. Conclusions The micropropagation of most conifers is often limited by the difficulty of rooting, and it was therefore essential to carry out an extensive bibliographic search on the subject before in vitro culture and biotization experiments. Based on information obtained from various sources, several physical and chemical factors were adjusted to improve in vitro rooting of P. pinea microshoots. An improvement in shoot rooting up to 70% was achieved for most of the tested clones (data published elsewhere). However, the roots obtained ceased growth. A new strategy was developed based on the co-cultivation of P. pinea and different ectomycorrhizal fungi (biotization). The methodology adopted was presented in this article. Biotization produced positive results, since they effectively contributed to overcoming the cessation of root growth and the improvement of various rooting parameters [9]. Methods and Protoc. 2018, 1, 34 9 of 10

In some cases, regenerated adventitious roots may develop mycorrhizal-like structures without the presence of fungus. There was a strong similarity between extensive dichotomous and coralloid branching of lateral roots that grew spontaneously in stone pine with those derived from fungal inoculation. Due to this similarity it may be difficult to diagnose ectomycorrhizas without confirmation of the ECM status by histological analysis. The appearance of mycorrhizal-like-structures depends on environmental conditions, the genetics of the species and the use of growth regulators. Abundant mycorrhizal-like structures in stone pine roots were produced by axenic cultures, in in vitro cultures, and in the subsequent acclimation phase in mixed substrates. More studies will be needed to elucidate the biochemical mediators that modify root architecture and its relationship with other compounds. Histological studies in combination with molecular techniques (the use of PCR-M13), allowed us to separate the different groups of fungi that can be found in stone pine forests in southern Portugal. In addition, it was possible to monitor mycorrhization stages.

Author Contributions: The authors have contributed in an equitable manner in the preparation of the manuscript. C.R.: development of protocol, preparation of the manuscript outlining, literature review, correction and suggestions in the text. M.A.Z.: preparation of the manuscript, bibliography update, improving the presentation and preparation of references. Funding: This work was supported by FCT Portugal: PTDC/AGR-CFL/71437/2006 Analysis and Mastering of Root Growth Signalling by Ectomycorrhizal Fungi on Pinus pinea Microshoot Cultures. Conflicts of Interest: The authors declare no conflict of interest.

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