Screening for Changes on Iris germanica L. Rhizomes Following Inoculation with Arbuscular Mycorrhiza Using Fourier Transform Infrared Spectroscopy

Abstract

Iris germanica L. is an ornamental and medicinal plant used since ancient times for their rhizomes, still utilized today to obtain orris butter highly valued in perfumery. Iris germanica presents special root adaptations, which confers certain tolerance to water and salt stress, making it a good option in the context of the current climate trend. Aim of this study was to prospect the potential for biofortification of rhizomes using commercial arbuscular mycorrhizae (AM) application in field conditions for six Iris germanica cultivars. Plants presented Paris-type AM colonization. Rhizome samples collected after nine months from treatment and maturated, presented FT-IR (fourier transform infrared spectroscopy) spectra variation between experimental variants. Presence of the main metabolites in rhizome could be confirmed based on literature. Screening focused on two rhizome quality markers: carbohydrates, which influence plant development, and fatty acids, which are extractable from rhizome. Results suggest potential to enhance their accumulation in certain cultivars, such as ‘Pinafore Pink’ following AM application.

1. Introduction

Iris germanica L. is a widespread ornamental plant [1] and has been used since ancient times in Europe for their rhizomes, currently highly valued in perfumery. Dry rhizome pieces are used as a spice, while rhizome extracts find application in the cosmetic industry and food industry as flavoring agents or in the production of alcoholic beverages such as gin [2]. Commonly cultivated are Iris germanica L. var. florentina and Iris pallida Lam [3,4] but almost any old garden bearded iris can be processed for this purpose with varying results [5]. Important Iris sp. crops are found in Italy, Morocco, China and India [3]. Iris sp. rhizomes harvested are either decorticated and slowly dried according to traditional method up to several years or sliced and subjected to biotechnological process for a fast-induced maturation [2,5,6]. Next, “aged” orris is milled and steam distillated to obtain orris butter rich in irone and myristic acid [3,4,7,8] sold at 14640€ for 1 kg [9].

Iris germanica is native to eastern Mediterranean region [2], and as adaptation to drought-prone habitats Iris germanica roots evolved multiseriate exodermis (MEX)—a trait within a suite of specializations which confers certain tolerance both to water and salt stress [10]. Since these are main challenges plants face within the current climate trend [11], ability to cope with such factors make this perennial a suitable option for smart landscaping as well as attractive niche crop. In addition, Iris germanica is a model plant for root studies [10], yet interaction with root fungal symbiotic colonizers and the outcome of this partnership has not been given much attention yet.

Arbuscular mycorrhizae (AM) is a symbiosis between plants roots and fungi from phylum Glomeromycota known for improving nutrition of plants, particularly in phosphorus [12]. Since phosphorus is required for biosynthesis of plant metabolites, its availability plays a crucial role [13]. Thus, common responses to AM may occur in relation with different plant metabolites, which are connected to general P-controlled pathways [14]. The positive influence of inoculation with arbuscular mycorrhizal fungi on certain plant metabolites [15,16] indicates that their application could be used as a strategy of crop biofortification [17] and many researches in the last decade aimed to identify specific effects AMF (arbuscular mycorrhizal fungi) can have on phytochemicals of economic interest [15,16,18,19,20]. Remarkably, many metabolite classes are affected by symbiosis with arbuscular mycorrhiza fungi species, and systemic responses have been found in various species from diverse plant families [14].

As a result, potential of AM to enhance phytochemical content attracted particular interest due to positive economic implications this might have, with wide range of researches conducted mostly in controlled environment. Review of over 40 representative studies from the last two decades, across ten botanic families in relation with changes in different metabolites from aromatic and medicinal plants due to AMF application, indicate that 37% of these studies were performed on plants from the Lamiaceae family. Across all ten botanic families, in over half of cases (65%) inoculated species were from two common Glomeraceae genera. On first place as most frequently used is genus Rhizophagus (45%) with Rhizophagus irregularis as the main species found across studies, followed by genus Funneliformis (20%) with Funneliformis mosseae as the most frequent species [21].

Fourier transform infrared spectroscopy (FT-IR) is a sensitive tool allowing measurement based on vibration of molecular bounds [22], which have specific frequency corresponding to an energy level. Spectra obtained are a graphical representation of absorption in units (A) versus IR frequency in terms of wavenumber (cm⁻¹). Bands in a spectrum are characterized by a wavenumber at which absorption occurs, corresponding to chemical bonds and the intensity of absorption, proportional to the amount of substance from sample [23]. Spectra are highly reproducible and informative representing signature of the overall chemical composition from a sample. FT-IR can be used both for identification purposes and for quantitative and qualitative analysis. In life sciences has been proven useful in analysis of microbial bioprocesses [22] as well as plant composition [24]. However, to the authors’ knowledge this method has not been utilized yet to screen for changes in plant following AM application.

Two important constituents of the Iris germanica rhizome: starch and myristic acid [25] were selected as quality markers and their spectral fingerprint was analyzed in this study. Irones are also valuable compounds but are not secondary metabolites of plant since they are absent from fresh rhizomes, instead they derive post-harvest from precursors [3] and are a marker for orris butter quality [2].

The aim of this study was to screen using FT-IR for variations in phytochemical content of rhizomes due to AM inoculation and cultivar for Iris germanica grown in field conditions.

To achieve this, three objectives were defined:

• Test potential of FT-IR to screen for variation in rhizome composition;
• Identify if rhizome quality markers display response associated with treatments;
• Define fingerprint ranges that could be used to confirm presence of main metabolites.

2. Materials and Methods

2.1. Location and Conditions

The experiment was established in Botanical Garden UASVM Cluj-Napoca Romania, on October 2016. Previous plant grown on plot was Dahlia hybrida. Soil characteristics of the experimental plot for topsoil (depth of 0–20 cm) were determined at an authorized pedological laboratory from Cluj-Napoca. Analysis is presented in

Table 1. Soil characteristics of the experimental plot [27,28].

Parameter	Method	Results
pH	Potentiometric	6.72
Humus	Walkley–Black	1.35%
Nitrogen	Kjeldahl	0.461%
Phosphorus	Colorimetric	68 ppm
Potassium	Flame photometry	312 ppm
Basic cation saturation	Kappen	20.96 me/100 g
Hydrolytic acidity	Extraction	1.92 me/100 g
Base saturation (V)	Calculation	92
Granulometric	Kacinscki	Coarse sand 14.42, fine sand 25.08, silt I 7.95, silt II 13.65, clay 38.90

, results indicated clay-loam soil type with low humus level, but good nitrogen, phosphorus and potassiumsupply according to thresholds from literature [26].

Climatic data for interval between October 2016–July 2017, corresponding to interval between planting and rhizome harvesting can be seen in Figure 1. During overwintering of the plants in field, temperatures reached as low as −18 °C in January and as high as 34 °C in July at the end of the experiment. Highest precipitation levels in descending order, were registered during months of October—prior and during planting—and in April when plants were in leaf elongation phenophase. Water availability at the time of planting ensured rooting of plants in autumn before winter dormancy and favorable growth conditions were met again in spring at when plants entered in vegetation.

2.2. Treatments

The experiment was organized according to bifactorial design: Factor A—Iris germanica cultivars with six levels: a₁ = ‘Black Dragon’ (black flowers), a₂ = ‘Blue Rhythm’ (blue flowers), a₃ = ‘Sultan’s Palace’ (red flowers), a₄ = ‘Lime Fizz’ (yellow flowers), a₅ = ‘Pinafore Pink’ (pink flowers) and a₆ = ‘Pure as The’ (white flowers) and Factor B—the treatment applied with two levels: b₁ = non-inoculated at planting (AM- and b₂ = inoculated at planting with 13 grams/rhizome commercial AM products (AM+).

Two products licensed by Royal Horticultural Society: “Bulb starter” and “After Plant” with root grow^TM mycorrhizal fungi and destined for outdoors and garden use, produced by company PlantWorks Ltd. United Kingdom [30] were mixed and applied together. One is specially designed for geophytes while other is suitable for perennials in general. Both contained propagules of five AMF species: Funneliformis mosseae, Funneliformis geosporus, Claroideoglomus claroideum, Rhizoglomus intraradices and Rhizoglomus microaggregatum, in an organo-mineral carrier. The carriers according to the label are represented by vermiculite for the first product and PAS100 certified recycled green compost for the second one, to which are added selected essential trace elements for supporting the establishment of AM symbiose in field. Plant material imported from Holland was purchased from company Anthesis International Bucharest. Prior to planting, all rhizomes had roots and leaves trimmed, were washed with chlorine solution, rinsed and planted with “hump” facing south. Between the non-inoculated and inoculated rows were ensured 7 m distance. From a combination of the two factors resulted 12 experimental variants, which were organized in randomized blocks with three replicates. In total were planted 108 rhizomes (Figure 2).

2.3. Assays

2.3.1. AM Analysis

At post-anthesis, on June 2017 and after 8 months from planting the rhizomes, root samples were collected and prepared for microscopy using ink-vinegar staining technique [31] followed by AM colonization evaluation through the Trouvelot method [32]. Indicators were obtained using Mycocalc [33]. Pearson correlogram for AM colonization parameters for both sets of plants was obtained with Past 3 [34], and are presented in Figure 3

2.3.2. FT-IR Analysis

In July 2017 (after nine months from planting) when plants entered summer dormancy, rhizome segments of similar age were collected from each experimental variant. Rhizomes were immediately peeled and aged by slow drying for a duration of 1.5 years (similar with the traditional method used for obtaining “white orris”). Voucher specimens are deposited at the Microbiology Department from Faculty of Agriculture UASVM Cluj-Napoca. Fourier transform infrared spectroscopy analysis was conducted in December 2018 at the Spectroscopy laboratory from Life Sciences Institute “King Michael I of Romania” from Cluj-Napoca. Pieces of dry and aged rhizomes were turned into powder using agate mortar and pestle. Then, calcinated 200 mg KBr was mixed with 3 mg rhizome (orris) powder for each experimental variant and placed in spectral pellet press chamber steel kit. Vitrification of pellets was obtained using Specac hydraulic press by applying pressure of 10t for 2 min. KBr pellet measurements were conducted with Fourier Transform Infrared Spectrometer Jasco FT/IR 4100, at scan range 4000–350 cm⁻¹, accumulation 256 and resolution 4.0 cm⁻¹. Spectra obtained was corrected for CO₂ and H₂O using Spectra Manager and further analysis conducted with Origin [35].

FT-IR spectra for rhizomes belonging to 12 experimental variants can be seen in Figure 4. These spectra correspond to rhizomes from Iris germanica plants, which benefited from application of the commercial products with AM (AM+) and control plants (AM–) of six cultivars. Changes that occurred for spectra refer to two types of characteristics: first, variations in peak position and secondly in peak intensity. These spectra features have been used before in FT-IR screening of rhizome samples belonging to Zingiberaceae species [36]. Shift in peak position refers to a displacement or change in peak wavenumber on FT-IR spectra for inoculated compared to non-inoculated plants of each cultivar, with a condition that the difference in positioning at either higher or lower wavenumber should be of at least 4 wavenumber units (cm⁻¹) and are marked on peak with a small pink or blue line (Figure 4). Increase or decrease in intensity is indicated by small arrows beneath bands screened for changes (Figure 4). In text when peak intensity changes are mentioned between non-inoculated versus inoculated plants, the values are provided, which represent how much the absorbance changed. Model peak analysis for two ranges proposed as marker bands for Iris germanica rhizome are detailed at the top of Figure 4, showing that contribution is given by different functional groups due to complex composition of rhizomes. List of peak readings for all 12 variants is provided in Supplementary Table S1.

3. Results and Discussion

3.1. Root AM Colonization

In June 2017 and after 8 months from planting in the field, AM colonization was observed for both sets of plants: inoculated and not inoculated. Proliferation pattern corresponded to the Paris morphotype with cell-to-cell spreading of hyphae and lack of intercellular spreading. Correlation between colonization parameters can be seen in Figure 3.

Arbuscules had the aspect of dense intra-cellular coils (Figure 1f,g). Comparing the arbuscules abundance—as most important indicators of symbiose activity between plant and fungi—on average were higher for inoculated plants. Thus, AM arbuscules abundance in root system of Iris germanica increased from A% = 5.26 in non-inoculated plants to A% = 7.14 in inoculated plants. In relative values this change equals to increase in the root system of 35.7%, while in mycorrhizal parts of root fragments (a%) the increase was of only 0.79% compared to non-inoculated plants. This corresponds to the mode of action of the inoculation products: following application of the products with AM fungi, plant roots met a higher density of AM propagules, which opened a higher number of colonization sites on the root system. For both sets of plants an almost perfect correlation (r² = 0.99) was found between intensity of colonization in roots fragments (m%) and root system (M%). A positive correlation was also found between these two intensity colonization indicators and arbuscules abundance in the root system for both sets of plants (Figure 3). Previous study reports that rhizomes of plants can also be colonized by AMF [37] but implications for plant are less clear.

3.2. Rhizome FT-IR Spectra

3.2.1. Starch Presence in Rhizomes

In Iris germanica, starch is the main reserve substance and accumulates in rhizome in high quantity, up to 50% [25]. Stored carbohydrate reserves in geophytes have influence on plant entrance in vegetation and is considered a quality marker due to direct influence on plant development [24]. An improved starch accumulation ensures adequate growth prior to flowering.

Starch possesses OH, C–H, C–O–C and C–O functional groups, which display typical peaks in FT-IR spectra across several ranges. Based on FT-IR analysis of starch from corn, cassava and potato [38], starch vibration modes in Iris germanica rhizome samples were also identified.

Thus, peaks between 763–770 cm⁻¹, 861–862 cm⁻¹ and 925–928 cm⁻¹ were characteristic to C–O–C ring vibration of starch molecule [38], while C–O–C asymmetric stretching vibration [38] was responsible for peaks occurring between 1154–1159 cm⁻¹ in Iris germanica samples. Strong peaks from ranges 1016–1020 cm⁻¹ and 1077–1078 cm⁻¹ were caused by C–O stretching vibration from starch [38]. Within a similar range, presence of carbohydrates caused peaks found at 1077 cm⁻¹ and 1146 cm⁻¹ in Curcuma caesia rhizome [39], and at 1018 cm⁻¹ in rhizome and roots of Panax ginseng [40].

Symmetric scissoring vibration of CH₂ from starch molecule is responsible for peak from range 1416–1428 cm⁻¹ of Iris germanica rhizome samples while symmetric deformation of same functional group [38] was causing the peak occurring between 1454–1458 cm⁻¹ (Figure 4). The peak observed at 1639–1648 cm⁻¹ was caused by C–O bending vibration associated with OH group from starch as shown by previous authors [38]. Starch C–H stretching vibration had a contribution to the band from ≈2930 while O–H stretching from starch molecule had contribution to band from ≈3440 [38], bands that were also present in Iris germanica rhizome samples (Figure 4).

Changes in relation with starch present in rhizome should be indicated by changes in position and intensity of characteristic peaks for starch identified. Rhizome samples of Iris germanica ‘Pinafore Pink’ from plants which benefited from AM products at planting presented increased intensity (with 0.15–0.41 absorbance increase) for typical starch peaks situated in the range identified above as well as for the two broad bands between 2800–3600 cm⁻¹. Similarly, Iris germanica ‘Pure As The’ also presented increase in peak intensity (with 0.01–0.23 absorbance increase) for all typical starch peaks with the exception of peak situated between 1639–1644 cm⁻¹. Samples from ‘Sultan’s Palace’ and ‘Lime Fizz’ AM+ plants, presented an increase in band intensity for the two broad bands situated between 2800 and 3600 cm⁻¹ to which starch vibration modes also had a contribution but not for all the other typical peaks. Based on these it was concluded that products with AM applied could have favored starch accumulation in rhizomes of ‘Pinafore Pink’ and to some degree to ‘Pure As The’. Previous studies in relation with effect of AM application on plant composition suggest that changes are due to improved nutrition following colonization [21]. This effect was not identified in all cultivars. An explanation could be the existence of specificity, which controls the outcome of AMF-plant interaction, as it was demonstrated for several medicinal plants, or changes might not be detectable [21]. In this regard, specific metabolite accumulation was conditioned by fungi species in Salvia sp. [41] and Zingiber sp. [42] and by cultivar for Mentha sp. [43]. The potential to increase starch accumulation and maximize beneficial effects for plant requires the identification and selection of genotypes, which exhibit the best response to AM inoculation in field.

3.2.2. Myristic Acid in Rhizomes

Myristic acid is the most abundant fatty acid in the Iris germanica rhizome [25], and is responsible for butter consistency of the commercial extract obtained from rhizomes [2]. It can be isolated and be used as raw material in the beauty industry [44], while methyl and ethyl esters of myristic acid are used as blenders in violet type perfume bases [45].

Typically, FT-IR myristic acid spectra shows distinctive peaks at 2848 cm⁻¹ and 2916 cm⁻¹ due to symmetrical and respectively asymmetrical stretching of CH₂, as well as –OH swinging or rocking vibrations within the range 680–940 cm⁻¹ due to aliphatic chain [46] but have weaker intensity_. In Iris germanica samples, peaks position from ≈2900 cm⁻¹ did not vary among cultivars but were situated at a higher wavenumber. The first one, was situated at 2855 cm⁻¹ and appeared more distinctive for ‘Pinafore Pink’ and ‘Pure As The’, which could indicate to a genotype particularity. The second one occurring in the spectral range 2926–2928 cm⁻¹ showed an increase intensity in samples from AM inoculated plants belonging to four cultivars, with exception of ‘Black Dragon’ and ‘Blue Rhythm’. Intensity change corresponded to a 0.12–0.37 absorbance increase. In a similar range, signal attributed to fatty acids was identified for Curcuma caesia rhizome samples at 2924 cm⁻¹ [39], for rhizomes and roots of Panax ginseng at 2923 cm⁻¹ [40] and in Curcuma longa rhizomes at 2925 cm⁻¹ [47].

3.2.3. Other Metabolites in Rhizomes

Presence of other major compounds in Iris germanica rhizomes was confirmed by comparison with previous studies conducted on geophyte plants.

A pervious study on roots of Iris germanica has put in evidence a distinctive peak at 1510 cm⁻¹ assigned to vibrations of molecular bounds from aromatic structure of lignin, which is less sensitive to IR and as consequence presents a weak intensity [48]. In rhizomes of Iris germanica this peak occurred between 1509 and 1516 cm⁻¹ and also has weak intensity. Interestingly, this peak is more distinguishable in samples of ‘Blue Rhythm’ and ‘Sultan’s Palace’ compared to other cultivars regardless of treatment, which could indicate to higher lignification of rhizome as the genotype characteristic. Lignin is essential for structural integrity of cell walls of vascular plants but is also a first line in defense against pathogens, lignification being linked to disease residence in plants [49]. Soft rot is a very widespread disease of cultivated Iris [1] and genotypes displaying this characteristic might prove helpful in breeding improved ornamental irises with rot-resistance. This aspect could be a premise for investigation in the future.

Phenols and proteins presence in rhizome could also be confirmed. Thus, the peak occurring within range 1369–1372 cm⁻¹ of Iris germanica samples, could be attributed to phenols, similar peak being identified previously in the Valeriana wallichii root at 1373.32 cm⁻¹ due to C–O stretching vibrations [50]. The peak found between 1639–1648 cm⁻¹ in Iris germanica rhizomes indicates to the presence of proteins as assigned by previous studies conducted on Curcuma longa rhizomes with the peak corresponding to amide I displayed at 1644 cm⁻¹ [47], and at 1640 cm⁻¹ or 1635 cm⁻¹ in Curcuma caesia rhizome [39,51].

Broad band between 3000 and 3600 cm⁻¹ showed high variation among cultivars for peak position, which situated between 3352 and 3417 cm⁻¹. For rhizome sample belonging to AM inoculated plants, it is noticeable an increase of overall band intensity in four cultivars. Vibrations of molecular bounds from several compounds contribute to this band [39,40]. However, previous authors, after studying Iris germanica root cells made the observation that a reduced protein content in sample results in a decrease of band intensity at 3320 cm⁻¹ [48]. Similarly, band between 1000 and 1200 cm⁻¹ besides the starch vibration modes already discussed, a contribution is made also by the CH₃ functional group [51] from the structure of cellulose [39].

Vibration modes of different functional groups give arise to several peaks of weaker intensity. The peak occurring at 702–706 cm⁻¹ in all Iris germanica rhizome samples are due to the vibration of molecular bounds from aromatic ring, as indicated by studies on rhizomes of Polygonum cuspidatum [52] and Hedychium coronarium [53]. Peak situated between 1240 and 1246 cm⁻¹ from Iris germanica samples can be attributed to C–O stretching vibrations, similarly described for Valeriana wallichii roots [50], while the one between 1311 and 1319 cm⁻¹ to the C–H functional group as identified in the Curcuma caesia rhizome [51]. The peak between 1730 and 1745 cm⁻¹ in Iris germanica rhizomes could be attributed to C=O stretching vibration, with peaks in similar range identified before in Iris germanica roots [48], rhizomes from species Curcuma longa, Curcuma xanthorrhiza, Zingiber cassumunar [36] and underground plant parts of Panax ginseng [40].

3.2.4. Significance of the Findings

Using FT-IR analysis to screen Iris germanica rhizome samples collected after nine months from AM application, presence of main compounds from rhizome was confirmed, as well as potential to improve quality for certain cultivars. Previous study showed that enhancement of secondary metabolites in licorice roots due to AM inoculation became evident after six months [54].

On an effect scale, ‘Pinafore Pink’ seemed to present favorable response, followed by ‘Pure As The’. Interestingly, as showed in previous works regarding vegetative [27] and physiological [28] parameters of these plants, emerges a similar pattern.

Difference due to the genotype response could be explained by referring to the genetic background of these tetraploid cultivated hybrids. Modern traits such as wide and ruffled petals controlled by recessive genes [1] can be admired in the more recent garden Iris category and it is the case for cultivars with red, pink, yellow and white flowers from this study. Intensification of color observed in the black-flowered cultivars of today is credited to genetic contribution of a wild species with dark flowers [1] thus, setting apart the cultivar ‘Black Dragon’ from the others. From cultivars studied here, oldest one is ‘Blue Rhythm’ [55], which could be considered closely related to historic cultivated forms since it lacks modern flower traits. Based on these it is suggested that genetic background might set apart cultivars also in terms of response to commercial AM products.

Previous authors remarked the current practical necessity of more AM experiments in the field and voiced existing difficulties in drawing clear evidence as prerequisite in justifying certain direction for agronomic practice [56]. Thus, experiments in real-life conditions can address practical challenges related to efficiency of microbial inoculants in agriculture.

4. Conclusions

FT-IR is a suitable technique for comparative characterization of plant constituents. Presence of main Iris germanica metabolites can be confirmed based on spectra analysis, including for rhizome quality markers such as carbohydrates and fatty acids.

Results suggest that application of commercial products with AM could enhance accumulation of starch and myristic acid in rhizome of certain cultivars such as ‘Pinafore Pink’. Thus, commercial products with AM could have a positive influence both on plant development as well as on extractable compounds.