A Polysaccharide-Based Integrated Nutrient Management System Enhances the Antioxidant Properties in Origanum dictamnus (Lamiaceae), a Valuable Local Endemic Plant of Crete

Abstract

Origanum dictamnus L. (Lamiaceae), a local endemic plant of Crete (Greece), creates polysaccharide-containing subcuticular compartments presenting biological activity against phytopathogenic fungi, and, among others, significantly affects the fungal cell wall polysaccharides. This field study introduces a fertilization scheme for O. dictamnus, which was developed and refined to optimize the yield as well as critical herbal quality aspects. Five fertilization schemes were investigated, based on a polysaccharide-based Integrated Nutrient Management (INM), a mixture of conventional inorganic fertilizers (ChF) and two biostimulants (not algae) via foliar and soil application. Plant growth, together with leaf chlorophyll fluorescence and color (SPAD meter, DA meter, Chroma Meter) were determined. The leaf content of chlorophyll, three critical antioxidant compounds (carotenoids, flavonoids, phenols) and nutrients were also assessed. Considering all three antioxidants together, the enhanced efficiency, non-toxic, water-soluble, polysaccharide-based INM by foliar application was the most stimulatory scheme, playing an important role in plant growth and development. The present field study provides, for the first time, baseline fertilization data improving key herbal quality features in O. dictamnus and unravels the attainment of high antioxidant properties. The latter may be exploited in favor of its further utilization as a raw material for tea preparation, medicinal purposes, natural food flavoring and/or food preservative.

1. Introduction

In modern times of global climate change challenging society and the conservation science, there are cumulative alarms surrounding the increasing human population and concerns on finding ways to enhance crop production to meet such demand [1,2]. Fertilizers are in authority for increasing crop yield by providing the essential nutrients for plants. On the other hand, the volatilization and leakage procedures have an impact on 30 to 50% loss of the applied fertilizers, causing severe pollution problems in rivers, lakes, waters, seawaters, and soil. Furthermore, to overcome such losses, the quantity of applied fertilizers is always greater than crop needs. New combinations of fertilizing materials have been planned to overcome these problems, namely Enhanced Efficiency Fertilizers (EEFs). The EEFs can regulate the accessibility of nutrients in the soil, decreasing nitrogen losses and pollution, so increasing crop yield for sustained food security without compromising human health and the environment [3,4].

Modern research in agriculture has developed distinctive groups of materials with innovative properties to improve agribusiness and increase crop production. Over the last 20 years, polysaccharide-based fertilizers have gathered significant consideration in the frame of EEFs. By being biodegradable, non-toxic, water-soluble, swellable, easily chemically modified and by increasing the disposal of nutrients, polysaccharide-based fertilizers are gaining a title role in agricultural practices [5]. Polysaccharides have further been used in several agricultural practices, such as in plant protection against biotic and abiotic stresses and in recovery of contaminated soil and water [6], in plant seed priming [7] and in improvement of plant product quality and bioactivity [8].

The Integrated Nutrient Management (INM) fertilization usually employs liquid polysaccharide-based fertilizers, further consisting of commercial compounds derived from chemical synthesis, systemic copper, biostimulants, organic calcium and urea as a nitrogen source. Plant biostimulants applied to plants comprise any material with the capacity to enhance nutrient use or stress toleration or to ameliorate quality features regardless of their composition. The polysaccharides usually include compounds with iron, aluminum, zinc, and copper, which makes them more easily available for plants. Amongst nutrients, copper is a critical micronutrient and its application together with polysaccharides has been well-established as beneficial for cultivation [9]. The INM is mainly based on organic edible raw materials and plant extracts, resulting in a full supplement for plants with amino acids, vitamins, sugars, and macro-microelements. A characteristic of INM is that, beyond its role as a simple plant nutrient provider, it also feeds the soil biota due to its content in polysaccharides. Energetic microbial flora, in turn, is closely associated with plant health, increased biomass, decreased nutrient leaking and higher nutrient availability. The microorganisms also mineralize organic materials, releasing elements such as nitrogen, phosphorus, sulphur, potassium and iron, and may enrich the soil with vitamins and antibiotics that are absorbed by crops. Furthermore, INM polysaccharides discharge and attach to clay particles, hence, together with microorganisms, contribute to the formation of stable soil aggregates. Such combinations may often lead to an early harvesting period, achieving the highest yield and quality of products; these are able to control the biological nitrogen transformations in soils and additionally may contribute to an exceptional strengthening of plants, which can become more resistant to cold and UV sunburns [10]. Numerous recent publications on polysaccharide-based INM fertilization schemes have shown to date that the paybacks of EEFs as a result of evaded social costs of nitrogen losses, may substantially overshadow their costs, and thus, they are highly recommended for integration in fertilizer strategies (e.g., [9,10,11,12,13]).

The fertilization formula sets not only the expected yield, but also defines product quality [14]. Quality is a broad term, encompassing several features of varying importance depending on the intended use [15,16]. In medicinal plants, the content of antioxidant compounds is traditionally a critical index of quality [17,18]. These secondary metabolite compounds are referred to as health promoters, holding both preventative and therapeutic action against a variety of diseases and disorders [19,20]. Carotenoids, flavonoids, and phenolics are common as well as major antioxidant compounds that exhibit a strong antioxidant ability [17,18]. Notably, organic fertilizers and INM strategies have been associated with increased antioxidant compound accumulation [19,21,22,23,24,25,26,27,28,29]. Organic fertilizers and INM strategies have also been suggested to act as a tool in promoting leaf chlorophyll concentration [30] that is tightly related to green coloration intensity, which in an alternative and widely used quality index of herbal material [15].

Plant products have been traditionally employed for healing and curing a variety of diseases and disorders in large parts of the world [31,32,33] and the use of medicinal-aromatic plants (MAPs) and concomitant natural products is also bursting in popularity in Western societies to date [34,35]. For instance, 400 MAPs are commercially traded for therapeutic purposes in India, while over 1500 MAPs are traded in Germany; in both countries and against expectations, over 93% of these volumes are still sourced from wild populations [35]. Due to this growing demand, genetic resources are inevitably depleted in many cases; however, this situation not only raises the risk of species extinction due to overexploitation but can also disturb the ecological balance at the ecosystem level [31,36]. Provided that alternative options are rather limited, the introduction of focal wild-growing species of commercial interest in agricultural settings is a sustainable remedy to overexploitation of wild genetic resources. To make effective such an endeavor, a well-documented initial plant material should be employed coupled with extant propagation protocols and cultivation guidelines applied in well-designed and well-managed cultivation systems.

Our previous studies report, for the first time, a valuation of the total phenolic and flavonoid contents verified in other local endemic plants of Crete such as Verbascum arcturus L. [26], Origanum microphyllum (Benth.) Vogel [27], Sideritis syriaca L. subsp. syriaca [28] and Carlina diae (Rech.f.) Meusel and A.Kástner [29] and they inspire the usage of fertilization in stimulating the herbal antioxidant outline devoid of compromised optical quality or yield. These studies represent the first-ever cultivations of exemplary neglected and underutilized local endemic species of Crete, outlining their potential economic value. Furthermore, our fertilization strategies represent multifaceted studies dealing with biological, physiological, phytochemical, and agronomical aspects of different plant species with promising potential in different economic sectors. Species-wise, the present investigation has focused on Origanum dictamnus L. (Lamiaceae), a perennial local endemic chasmophyte of Crete (Greece) threatened with extinction, i.e., assessed as Endangered [37], Vulnerable [38], or Near Threatened according to Flora of Greece web (https://portal.cybertaxonomy.org/flora-greece/cdm_dataportal/taxon/11dfbc28-3e28-4187-bce3-ac6178bee770, accessed 30 November 2023). O. dictamnus L. is traditionally cultivated at a local scale in Crete as one of the most popular and therapeutic aromatic-medicinal plants, directly sourced from the wild since ancient times and strongly associated with the Cretan cultural heritage [39,40] as a contemporary yet traditional herbal medicinal product widely used for tea preparation, medicinal purposes, natural food flavoring and/or food preservative [40,41]. In addition, its therapeutic indications are approved by the European Medicines Agency (https://www.ema.europa.eu/en/medicines/herbal/origani-dictamni-herba, accessed 30 November 2023). The O. dictamnus L. extracts possess high scavenging capacities, high phenolic contents and are rich in flavonoids [42], with benzoic acid and rosmarinic acid showing the highest concentrations [43]. Furthermore, O. dictamnus is known to produce polysaccharide-containing subcuticular compartments and possesses biological activity against phytopathogenic fungi, significantly affecting among others, the fungal cell wall polysaccharides [44].

During plant development, a polysaccharide stock is vital, as polysaccharides act as a direct energy source helping plants to avoid and adapt to stressful conditions. In this regard, the polysaccharide INM might activate several metabolic processes inside plant tissues, such as the elevated hydrolysis of the unique species-specific endogenous subcuticular-located polysaccharides and/or the hydrolysis of the exogenously added polysaccharides provided by INM application. As suggested by other studies, the latter, in turn, may eventually lead to the increase in the contents of physiologically active sugars, thus providing adequate biological resources for the synthesis of complex essential metabolic substances.

Due to limited relevant data on range-restricted and threatened plant species cultivated for the first time or only locally, the data generated from the experiments of O. dictamnus may enable more accurate comparisons to previously published data on other local endemic plants of Crete, such as V. arcturus [26], O. microphyllum [27], S. syriaca subsp. syriaca [28] or C. diae [29], thus facilitating better results’ interpretation. Context wise, the present study comparatively investigated the effects of different fertilization treatments and application techniques in a pilot field cultivation of O. dictamnus in Crete, contrasting a polysaccharide-based INM fertilization protocol with other schemes. The developed protocol responds to the extant information gap regarding the fertilization requirements of O. dictamnus and was specifically designed to improve its yield, as well as critical quality aspects (i.e., color intensity, antioxidant element concentration). In addition, this investigation explored nutrients’ accumulation and partitioning in different plant organs of O. dictamnus, for which only little is regrettably known [45]. Such knowledge would be essential to induce growth and biomass yield potential of O. dictamnus by regulating nutrients supply according to species-specific needs, further elucidating how the latter correlates with its physiological characteristics, oil production and antioxidant activity [46]. This research complements similar investigations [12] or other research lines related to the herein focal species (e.g., [47,48]), and is scoped to contribute to bridging the way towards the effective sustainable exploitation of such valuable Mediterranean genetic resources.

2. Materials and Methods

2.1. Focal Species and Origin of Plant Material

Dittany of Crete is a mat-forming and white-woolly chasmophyte 20–30 cm in height, with rounded leaves of velvety texture and lilac flowers bordered by purple-pink bracts in summer [49]. O. dictamnus is a seasonally dimorphic plant having a different appearance in winter and summer [50]. Shoots of late winter plants are almost leaf naked except for their apical region which bears a cluster of small leaves covered with a thick indumentum of non-glandular hairs (Figure 1). Shoots of summer plants are vigorous with large green leaves. Non-glandular hairs are dendroid with a five-celled vertical stub and several lateral branches. Glandular hairs are of two types, large peltate hairs and small capitate hairs. Peltate hairs are numerous and consist of a 12-celled head, a unicellular stalk, and a basal epidermal cell. Peltate hairs secrete an essential oil, high in carvacrol content. Peltate glandular hairs of O. dictamnus create at the stage of secretion two subcuticular chambers, one large and bladder-like at the apex of the head (containing essential oil), and a small and ring-like one at the bottom of the head (containing polysaccharides). In the apical chamber containing the essential oil, a small lateral compartment is present comprising polysaccharides. The secretory product occurs in the form of glucosides that pass through the plasmalemma to enter the apical chamber which are hydrolyzed to the aglycone fraction (essential oil) and the sugar fraction (polysaccharides) [51].

Botanical expeditions were performed to explore different areas for wild-growing populations of O. dictamnus along gorges and mountain plateaus of Crete, Greece (Figure 1) in the frame of the research project “Conservation and sustainable utilization of rare-threatened endemic plants of Crete for the development of new products with innovative precision fertilization” (acronym: PRECISE-M, Τ1ΕΔΚ-05380). Seed collections were performed using an authorized special permit of the Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization Demeter (Permit 82336/879 of 18 May 2019 and 26895/1527, 21 April 2021) which is issued yearly by the Greek Ministry of Environment and Energy.

The collected seeds and voucher samples were taxonomically identified, and unique IPEN (International Plant Exchange Network) accession numbers were assigned to each independent collection (GR-1-BBGK-03,2108; GR-1-BBGK-19,330; GR-1-BBGK-21,91) by the Balkan Botanic Garden of Kroussia, Institute of Plant Breeding and Genetic Resources (IPBGR) of the Hellenic Agricultural Organization, Demeter (ELGO Dimitra). New plants were initially raised ex situ for further field experimentation [47]. The plants used in the experimental procedure were transplanted in 2 L plastic pots by the company of AFI GLAVAKI KE SIA OE Tree and Plant Nurseries, Aridea, PELLAS, GR-58400, Greece.

2.2. Field Experiment

The field experiment was performed at the farm of the Hellenic Mediterranean University, Heraklion, Crete (Greece) in a research field of 20 m × 25 m (35°19′ N, 25°6′ E, 60 m). The plants were established on the ground on 1 March 2021. The distance between plants was 40 cm, and between rows was 80 cm. The rows were 20 m long (Figure 1). These rows had east–west direction and included as “guard plants” were other local Cretan endemics, i.e., individuals of Sideritis syriaca subsp. syriaca (Malotira or Cretan Mountain tea [28]), Origanum microphyllum (Cretan marjoram, [27]), Carlina diae (Carline of Dia [29]), and Verbascum arcturus (Cretan mullein, [26]), all investigated in the frame of the research project PRECISE-M. Each of the above plant species primarily aimed to identify species-specific responses to the fertilization schemes applied, thus testing the same fertilization treatments in terms of yield and quality characteristics in different biological species (“biological replicates at species level”).

The experimental design was performed in completely randomized blocks of 10 plants of O. dictamnus per block, three blocks per treatment (see below), which were randomly located in different rows (Figure 1C). To achieve the same starting plant material, all plants were trimmed at 5 cm above ground level at the end of April. At the end of May (i.e., 30 d following trimming), six fertilization treatments were initiated which were weekly performed till the final harvest. An automatic irrigation system was constructed with 2 L h⁻¹ adjustable drippers to supply water to the plants three times per week. Pest and disease control was not necessary, but removal of weeds was regularly required, and it was manually performed. The final harvest was carried out on 30 June 2021.

The soil properties of the research field are reported in previous publications [26,27].

2.3. Fertilization Regimes

The pilot cultivation of O. dictamnus followed weekly treatments using water (control), conventional inorganic fertilization (ChF), biostimulant and INM in liquid or soluble granule form administered with foliar and soil applications. The foliar applications were performed using a 5 L plastic handheld sprayer (low pressure) until apparent wetness, and the soil applications were manually performed (100 mL nutrient solution per plant). The INM and biostimulants were supplied with four special fertilizers from THEOFRASTOS company, Industrial area of Korinthos, GR-20100 Korinthos, Greece. These polysaccharide-based semi-organic fertilizers are made from high quality organic edible raw materials and plant extracts, resulting in a full supplement of plants with amino acids, vitamins, sugars, and macro-microelements. The INM polysaccharides are aimed to contribute to the formation of stable soil aggregates formed in connection with active soil microorganisms; INM polysaccharides when foliar sprayed, they are expected to be rapidly absorbed by the plants. More specifically: THEORUN is a nitrogen source liquid fertilizer with N 17% (w/w), P₂O₅ 0% (w/w). K₂O 1.5% (w/w), organic matter 3.2% (w/w), C/N 0.09, pH 9.1, diurea 0.26%, electric conductivity 86 mS/cm [liquid extract (1‰)]. THEOCAL is an organic calcium powder fertilizer with raw materials based on calcium formate molecules, pH 7.1, Ca 30% (w/w) and organic matter 30%. THEOFAST is a liquid biostimulant made from plant extracts of edible raw materials (not algae) with organic matter 4.4% (w/w) (plant extracts), pH 9.5 and electric conductivity of 78 mS/cm [liquid extract (1‰)]. THEOMASS is a liquid biostimulant including plant extracts of edible raw materials (not algae) with organic matter 5.4% (w/w) (plant extracts), pH 9.4 and electric conductivity 85 mS/cm [liquid extract (1‰)]. The chemical fertilizers were all in soluble powder or granule form, except the liquid fertilizer for micronutrients (Plex Mix, AGRI.FE.M. LTD Fertilizers, Aspropirgos, Greece). Two foliar fertilization treatments (INM and ChF), three soil application treatments (INM, ChF and a biostimulator) and a control for the pilot cultivation of O. dictamnus in the field were employed in the experimentation (Supplementary Material Methods S1).

2.4. Measurements of Plant Features

Several plant and leaf level measurements were regularly conducted. Among replicate plant individuals, sampling was randomly conducted. Leaf coloration and chlorophyll fluorescence were assessed at vegetative, early flowering, and full flowering stages, whereas the remaining measurements were targeted only to the full flowering stage. For the full flowering stage, non-invasive measurements were conducted 2–5 d prior to the destructive harvest (i.e., 30 June 2021). For leaf level measurements, fully expanded leaves were selected from the upper (towards the apex) one third leaf-bearing nodes of the stem which developed under direct natural light. In all instances, the time between sampling and the initiation of the evaluation was shorter than 15 min. When was necessary, leaf samples were placed in vials, flash-frozen in liquid nitrogen and were transferred to a freezer (−80 °C) for storage. Replicate leaves were sampled from separate plant individuals.

2.4.1. Non-Destructive Evaluation of Leaf Coloration at Three Growth Stages

Leaf coloration depends on the fertilization regime, and affects both photosynthetic capacity and visually perceived quality [52]. In this study, it was assessed by three different methods as follows:

(i)

Leaf SPAD value as a proxy of chlorophyll content was determined by using a SPAD-502 (Konica Minolta Corp., Solna, Sweden).

(ii)

The index of absorbance difference (IAD) accurately evaluating fruit ripeness, since it is closely associated with outer mesocarp chlorophyll content and IAD was computed as the difference between the absorbance values at 670 and 720 nm, near the chlorophyll absorbance peak [26]. The potential of IAD in reflecting respective differences in leaves has not been previously evaluated. In this investigation, IAD was determined in leaves by using the DA meter (tr DA Meter, T.R. Turoni, Italy) [53].

(iii)

Leaf color was quantified by using a Chroma Meter (Model CR-400, Minolta Corp., Tokyo, Japan). CIE L*a*b* coordinates were recorded using D65 illuminants and a 10° Standard Observer as a reference system. L* [a measure of lightness, ranging from 0 (black) to 100 (white)], a* (a measure of intensity in the green to red range, where negative values refer to green and positive to red), and b* (a measure of representing intensity in the blue to yellow range, where negative values refer to blue and positive to yellow) were obtained [28].

These measurements were in situ conducted in attached leaves of intact plant individuals during vegetative, early flowering, and full flowering stages. Three points were recorded per replicate leaf and were further averaged. Three replicate leaves were assessed per treatment.

2.4.2. Non-Destructive Evaluation of Photosynthetic Performance at Three Growth Stages

The ratio of variable to maximum chlorophyll fluorescence (Fv/Fm) was assessed as a valid indicator of leaf photosynthetic performance [54]. Measurements were performed by using a chlorophyll fluorometer (OS-30P, Opti Sciences, Hudson, NH, USA). Prior to evaluation, sampled leaves were dark adapted (≥20 min) by employing leaf clips. Fv/Fm was assessed by applying a saturated photosynthetic photon flux density of 3000 µmol m⁻² s⁻¹.

These measurements were in situ conducted in attached leaves of intact plant individuals at vegetative, early flowering, and full flowering stages. Three points were recorded per replicate leaf and were further averaged. Three replicate leaves were assessed per treatment.

2.4.3. Leaf Shape Indicators

A morphometric analysis was performed by analyzing leaf form. Leaf shape traits were derived from images acquired by a digital camera employing a copy stand (Sony DSC-W830, Sony Corporation, Tokyo, Japan) that was adjusted to 0.5 m away from the subject under non-reflective glass. Using specialized software (ImageJ, version 1.54h; Wayne Rasband/NIH, Bethesda, MD, USA), leaf lamina outlines were processed to estimate the following four (dimensionless) metrics of leaf form: (a) aspect ratio [(major axis)/(minor axis); axes of the best-fitted ellipse], (b) circularity [(4π × area)/(perimeter)²], (c) roundness [(4 × area)/[4π × (major axis)²]], and (d) solidity [(area)/(convex area)] [55]. Each metric quantifies a distinct feature of leaf shape. Aspect ratio and roundness are affected by the length to width ratio, while circularity and solidity are sensitive to serration and lobing. Aspect ratio ranges from 1 (circle) to value without upper bound (infinitely narrow). Roundness ranges from 0 (infinitely narrow) to 1 (circle). Circularity ranges from 0 (infinitely narrow) to 1 (circle). Solidity ranges from 0 to 1, being inversely related to boundary irregularities. Solidity is sensitive to leaves with deep lobes or a distinct petiole and can be employed to detect leaves lacking such structures. Solidity, unlike circularity, is not greatly influenced by serrations and minor lobing [56]. Thirty leaves (5 per plant × 6 plant individuals) were analyzed per treatment.

2.4.4. Plant Growth and Biomass Partitioning to Generative Organs

Plant growth and biomass partitioning to generative organs were determined. Above ground plant and inflorescence (fresh and dry) masses were determined (±0.01 g; MXX-412; Denver Instruments, Bohemia, NY, USA). For measuring dry weight, the samples were placed in a forced air drying oven for 72 h at 80 °C. Six replicate plants were evaluated per treatment.

2.4.5. Leaf Chlorophyll and Carotenoid Contents

The effect of growth conditions on chlorophyll and carotenoid contents was assessed. Following fine chopping, portions (0.5 g) were homogenized with the addition of 10 mL of 80% acetone. This primary acetone extract was then filtered, and the filtered extract was diluted by adding 2 mL of 80% acetone per mL of extract. Since chlorophyll is light sensitive, extraction took place in a dark room [57]. The obtained extract was subjected to reading on a spectrophotometer (Mapada UV-1800, Mapada Instruments Co., Ltd., Shanghai, China). Total chlorophyll and carotenoid contents were calculated [57]. Three leaves were assessed per treatment and for each replicate, four samples (collected from different plant individuals) were pooled, and the assay was performed twice.

2.4.6. Leaf Total Phenolic and Total Flavonoid Contents

Leaf samples (0.1 g) were extracted with 1 mL of 80% aqueous methanol in an ultrasonic bath (10 min) and were then centrifuged (15,000× g for 10 min). The contents of total phenolic and total flavonoid were determined by using the Folin–Ciocalteu assay and aluminum chloride colorimetric assay, respectively. The absorbance against prepared reagent blank was determined using a microplate reader (Infinite 200 PRO, TECAN Trading AG, Männedorf, Switzerland). For total phenolic content, gallic acid was used as the standard reference and gallic acid equivalent (GAE) was expressed as mg per g of fresh mass. For total flavonoid content, rutin was used as the standard reference and rutin equivalent (RUE) was expressed as mg per g of fresh mass. Three leaves were assessed per treatment. For each replicate, four samples (collected from different plant individuals) were pooled, and the assay was performed twice.

2.4.7. Leaf-Soluble Sugar Content

To assess the effect of the fertilization scheme on carbohydrate status, leaf samples (0.1 g) were incubated with 1 mL deionized water in a water bath (100 °C for 30 min). The homogenate was centrifuged (15,000× g for 15 min) at room temperature (25 °C). Then, 0.1 mL of the solution was mixed with anthranone ethyl acetate and sulphuric acid. Soluble sugar content was assayed in the supernatant by measuring the absorbance at 630 nm, using a spectrophotometer (Mapada UV-1800, Mapada Instruments Co., Ltd., Shanghai, China). Soluble sugar content was expressed on a fresh weight basis (mg g⁻¹). These measurements were conducted on three replicates per treatment. For each replicate, four samples (collected from different plant individuals) were pooled, and the assay was performed twice.

2.4.8. Leaf and Inflorescence Nutrient Analysis

To assess the role of fertilization schemes on nutrient absorption, leaf and inflorescence nutrient analysis was conducted. Samples were washed with distilled water and then dried. The biomass was dried at 70 °C, weighed, ground, and then analyzed for total N by the Kjeldahl method. In addition, sub-samples were ash-burned at 500 °C for at least 4 h; the ash was then dissolved using a 2 M HCl solution, filtered, and P, K, Ca, Mg, Cu, Zn, Fe, Mn and B contents were determined in the filtrate, employing flame photometry, atomic absorption spectrometry and UV–Vis spectrometry, depending on the element. Nutrient content was expressed on a dry weight basis. Three replicates were evaluated per treatment. For each replicate, four samples (collected from different plant individuals) were pooled, and the assay was performed twice.

2.5. Statistical Analysis

Data analysis was carried out using the SAS statistical software (version 9.4M7, SAS Institute, Cary, NC, USA). Data were tested for homogeneity of variances (Duncan’s test). Analysis of variance (one-way ANOVA) was conducted on the data and subsequently, the least significant differences (LSD) test was used for mean comparisons (p = 0.05).

3. Results

3.1. The Fertilization Scheme Exerted Limited Effects on Leaf Color

Leaf color is an important aspect of herbal quality, and therefore it was evaluated at three growth stages (i.e., vegetative, early flowering, and full flowering) by employing three methodologies (SPAD meter, DA meter, Chroma Meter). At the vegetative stage, plants receiving the INM-sa fertilization scheme presented a higher leaf SPAD value as compared to control (C) plants (Figure 2A). At the early and full flowering stages, the leaf SPAD value was not affected by the fertilization treatment (Figure 2B,C). At the vegetative stage, control (C) plants and plants under ChF-sa presented the lowest IAD value (i.e., being greener) as compared to the rest of the treatments (Supplementary Figure S1A). At the vegetative stage, plants under INM-sa showed the highest IAD value from all treatments besides INM-fa (Supplementary Figure S1A). At the early flowering stage, plants receiving INM-sa showed higher IAD value than control (C) plants, whereas plants receiving INM-fa exhibited lower IAD value than the controls (Supplementary Figure S1B). At the full flowering stage, the leaf IAD value was not affected by the fertilization scheme (Supplementary Figure S1C).

At the vegetative stage, plants receiving INM-fa had a higher leaf L value as compared to the remaining treatments (Supplementary Figure S2A). At the early flowering stage, plants receiving INM-fa presented a higher leaf L value as compared to control plants and plants receiving ChF-sa or MPE-sa (Supplementary Figure S2B). At the full flowering stage, control (C) plants and plants receiving both foliar fertilization schemes (INM-fa or ChF-fa) exhibited a higher leaf L value as compared to those under ChF-sa or MPE-sa (Supplementary Figure S2C). At the vegetative stage, plants receiving INM-fa showed a higher leaf a* value as compared to the remaining treatments (Supplementary Figure S3A). At early and full flowering stages, the leaf a* value was not affected by the fertilization treatment (Supplementary Figure S3B,C). At all three growth stages, the leaf b* value was not affected by the fertilization scheme (Supplementary Figure S4).

3.2. The Fertilization Scheme Did Not Affect Leaf Photosynthetic Performance

The effect of the fertilization scheme through different (foliar/soil) application methods on the chlorophyll fluorescence value of O. dictamnus at vegetative, early flowering and full flowering stages is shown in Figure 3. At all three growth stages, the leaf Fv/Fm value was not significantly affected by the fertilization scheme (Figure 3).

3.3. Fertilization-Induced Minor Effects on Leaf Shape

To evaluate the effect of the fertilization scheme on leaf form, four shape indicators (aspect ratio, circularity, roundness, solidity) were analyzed. Plants under MPE-sa showed lower circularity as compared to the rest of the treatments including the control (C) (Figure 4B). As far as the other three shape indicators are concerned, they remained unchanged upon fertilization treatment, since in all cases of the fertilization scheme and application method, they were similar to the control (C) (Figure 4A,C,D).

3.4. Fertilization Did Not Stimulate Plant Growth but Affected Biomass Allocation

Biomass accumulation, as related to the above-ground dry mass (Figure 5A) and tissue water content (Figure 5B) were not affected by the fertilization scheme. Plants receiving INM-sa presented the lowest partitioning to generative organs as compared to the remaining treatments including control (C) plants (Figure 5C). Plants receiving MPE-sa or INM-sa showed lower partitioning to generative organs as compared to the ones receiving ChF-fa (Figure 5C).

3.5. Fertilization Stimulated Leaf Chlorophyll Content and Affected Leaf Antioxidant Substances’ Content

As compared to the control (C) plants, fertilization stimulated leaf chlorophyll content in all cases, besides ChF-fa (Figure 6A). The highest leaf chlorophyll content was noted in plants treated with ChF-sa, followed by the ones that received INM-fa (Figure 6A). Plants receiving INM-fa, ChF-sa or MPE-sa showed higher leaf carotenoid content, as compared to control (C) plants (Figure 6B). Plants receiving INM-fa or ChF-sa exhibited the highest leaf carotenoid content, as compared to all of the remaining treatments (Figure 6B). Plants receiving INM-fa, ChF-fa or INM-sa showed higher leaf total phenolic content as compared to C and plants receiving ChF-fa had the highest leaf total phenolic content as compared to all of the remaining treatments (Figure 6C). Plants receiving INM-fa or ChF-sa had higher leaf total flavonoid content as compared to control (C) plants, with those receiving INM-fa showing the highest leaf total flavonoid content over all treatments (Figure 6D). Based on the aforementioned results, it seems that INM-fa presents the best fertilization scheme in antioxidant and quality terms.

3.6. Fertilization Treatments Generally Stimulated Leaf-Soluble Sugar Content

The effect of the fertilization scheme through different (foliar/soil) application methods on the leaf-soluble sugar content of O. dictamnus is presented in Figure 7. As compared to control (C) plants, the fertilization stimulated leaf-soluble sugar content in all cases, besides INM-fa (Figure 7).

3.7. Fertilization Treatment Affected Plant Nutrient Content

Leaf macro- and micro-nutrient content of O. dictamnus varied depending on the treatment and nutrient (

Table 1. Effect of the fertilization scheme through different (foliar/soil) application methods on leaf essential macronutrient content of Origanum dictamnus. C: control (water); INM-fa: Integrated nutrient management (INM) by foliar application; ChF-fa: conventional inorganic fertilization by foliar application; INM-sa: INM by soil application; ChF-sa: ChF by soil application; and MPE-sa: biostimulant by soil application (THEOMASS). Values represent the mean of three replicates ± SEM.

Treatment	N	P	K	Ca	Mg
			(g kg–1)
C	15.7± 0.4 bc *	1.7 ± 0.1 c	15.9 ± 1.1 bc	38.9 ± 0.5 ab	3.4 ± 0.0 a
INM-fa	18.7 ± 0.1 ab	4.9 ± 0.2 a	17.7 ± 0.4 abc	32.8 ± 1.1 bc	2.9 ± 0.0 bc
ChF-fa	18.8 ± 0.4 ab	4.9 ± 0.1 a	20.2 ± 0.8 ab	29.6 ± 1.2 c	3.4 ± 0.1 a
INM-sa	14.7 ± 0.7 c	3.2 ± 0.1 b	15.4 ± 0.3 cd	41.1 ± 1.0 a	3.3 ± 0.0 ab
ChF-sa	20.8 ± 0.5 a	3.9 ± 0.1 b	20.3 ± 0.5 a	37.0 ± 0.8 ab	3.4 ± 0.1 a
MPE-sa	14.0 ± 0.3 c	1.6 ± 0.0 c	11.1 ± 0.3 d	36.7 ± 1.0 ab	2.7 ± 0.1 c
p F-test	<0.001	<0.001	0.001	0.020	0.013

* Means followed by different letters within the same column are statistically different using the LSD test at p ≤ 0.05.

and

Table 2. Effect of fertilization scheme through different (foliar/soil) application methods on leaf essential micronutrient content of Origanum dictamnus. C: control (water); INM-fa: Integrated nutrient management (INM) by foliar application; ChF-fa: conventional inorganic fertilization by foliar application; INM-sa: INM by soil application; ChF-sa: ChF by soil application; and MPE-sa: biostimulant by soil application (THEOMASS). Values represent the mean of three replicates ± SEM.

Treatment	Cu	Zn	Fe	Mn	B
			(mg kg–1)
C	14.5 ± 0.5 cd *	33.5 ± 0.6 cd	1732 ± 48 b	79 ± 5 de	36.3 ± 0.4 c
INM-fa	20.4 ± 0.4 a	53.1 ± 2.2 a	1916 ± 73 a	210 ± 12 a	51.4 ± 1.0 a
ChF-fa	18.7 ± 0.5 ab	47.6 ± 1.6 ab	2009 ± 94 a	174 ± 7 ab	42.6 ± 0.8 b
INM-sa	15.6 ± 0.3 bcd	33.0 ± 0.3 d	1691 ± 49 b	118 ± 4 cd	38.6 ±0.7 bc
ChF-sa	17.7 ± 0.6 abc	42.4 ± 1.4 bc	1528 ± 44 c	132 ± 6 bc	42.5 ± 0.7 b
MPE-sa	12.5 ± 0.5 d	33.1 ± 0.7 d	1236 ± 62 d	60 ± 2 e	28.8 ± 0.6 d
p F-test	<0.001	<0.001	0.022	<0.001	<0.001

* Means followed by different letters within the same column are statistically different using the LSD test at p ≤ 0.05.

). Conventional inorganic fertilization applied through soil (ChF-sa) showed the highest values with respect to N and K content as compared to all other treatments including the control (C); the same stands for the INM-fa and ChF-fa treatments in the case of P (Table 1). Although significant differences were observed among treatments in respect to Ca and Mg content, all fertilization treatments showed similar or even lower content, in certain cases, in comparison to the control (C) (Table 1). As far as the micro-nutrient content is concerned, in all cases, the highest values were determined in the leaves of plants receiving INM-fa followed by plants receiving ChF-fa, compared to all other treatments including the control (C) (Table 2). It is worth noting that in most cases, MPE-sa showed the lowest content of both macro- and micro-nutrients in comparison to the remaining fertilization treatments. Moreover, in certain cases the leaf nutrient content observed with MPE-sa was even lower than the control (C) (Table 1 and Table 2).

The effect of fertilization treatment on inflorescence nutrient content was rather poor, since although different trends were obtained upon the fertilization scheme and application method, significant differences were obtained only for P from the macro-nutrients and Fe from the micro-nutrients (

Table 3. Effect of fertilization scheme through different (foliar/soil) application methods on floral essential macronutrient content of Origanum dictamnus. C: control (water); INM-fa: Integrated nutrient management (INM) by foliar application; ChF-fa: conventional inorganic fertilization by foliar application; INM-sa: INM by soil application; ChF-sa: ChF by soil application; and MPE-sa: biostimulant by soil application (THEOMASS). Values represent the mean of three replicates ± SEM.

Treatment	N	P	K	Ca	Mg
			(g kg–1)
C	10.54 ± 0.4 a *	1.70 ± 0.1 b	13.74 ± 0.6 a	16.31 ± 0.4 a	1.92 ± 0.1 a
INM-fa	11.85 ± 0.7 a	1.83 ± 0.1 b	15.49 ± 0.5 a	17.00 ± 0.9 a	1.70 ± 0.2 a
ChF-fa	12.84 ± 0.3 a	2.24 ± 0.2 a	15.45 ± 1.5 a	15.91 ± 0.1 a	1.90 ± 0.1 a
INM-sa	12.58 ± 0.8 a	1.35 ± 0.1 c	13.07 ± 0.9 a	16.39 ± 1.6 a	1.50 ± 0.1 a
ChF-sa	10.53 ± 1.6 a	1.69 ± 0.2 b	17.31 ± 1.5 a	15.98 ± 1.5 a	1.46 ± 0.2 a
MPE-sa	9.78 ± 0.6 a	1.48 ± 0.1 b	13.38 ± 1.6 a	16.00 ± 0.7 a	1.40 ± 0.1 a
p F-test	# NS	0.003	NS	NS	NS

* Means followed by different letters within the same column are statistically different using the LSD test at p ≤ 0.05. ^# Non-significant.

and

Table 4. Effect of fertilization scheme through different (foliar/soil) application methods on floral essential micronutrient content of Origanum dictamnus. C: control (water); INM-fa: Integrated nutrient management (INM) by foliar application; ChF-fa: conventional inorganic fertilization by foliar application; INM-sa: INM by soil application; ChF-sa: ChF by soil application; and MPE-sa: biostimulant by soil application (THEOMASS). Values represent the mean of three replicates ± SEM.

Treatment	Zn	Fe	B
		(mg kg−1)
C	12.97 ± 1.4 a *	21.88 ± 1.4 a	222.79 ± 5.1 a
INM-fa	14.68 ± 2.8 a	20.25 ± 1.1 a	322.93 ± 18.8 a
ChF-fa	15.57 ± 0.5 a	21.95 ± 0.6 a	270.11 ± 19.9 a
INM-sa	14.31 ± 0.4 a	12.61 ± 1.2 b	241.95 ± 40.8 a
ChF-sa	15.42 ± 1.0 a	15.03 ± 2.2 b	204.19 ± 35.3 a
MPE-sa	15.73 ± 0.4 a	14.49 ± 0.6 b	225.95 ± 37.4 a
p F-test	# NS	0.001	NS

* Means followed by different letters within the same column are statistically different using the LSD test at p ≤ 0.05. ^# Non-significant.

). More specifically, overall treatments including the control (C), ChF-fa showed the highest P content and INM-sa the lowest one (Table 3). Regarding the inflorescences’ micronutrient content in O. dictamnus, soil application (“sa”) of all fertilization schemes showed lower Fe content than the foliar (“fa”) application of both INM and ChF treatments, which were also similar to the control (C) (Table 4).

4. Discussion

The ongoing global interest on medicinal–aromatic plants adds increasing pressure on the wild-growing genetic resources and most of the traded herbal material is still directly collected from nature [31,36]. In this way, wild plant resources deplete at an alarming rate [58], threatening a wide range of species with extinction [54,55]. These changes may disturb the niche structure and balance, with adverse effects on the ecosystems and eventually human well-being [58,59,60].

Plant species conservation necessitates the introduction of commercially valuable taxa into agricultural settings and their sustainable utilization to alleviate the over-harvesting pressure on wild populations [52]. In response to this need, cultivation practices and farming operations ought to be developed and established, predominantly employing an optimized fertilization program. The field investigation herein presents the first comprehensive steps of introducing the threatened Cretan endemic O. dictamnus into sustainable cultivation and evaluates a fertilization scheme which is best for stimulating crop yield as well as two key features of herbal material quality. Under this background, polysaccharide-based INM fertilizers were also tested owing not only to the reduced environmental footprint, but also to a powerful fertilization scheme leading to improved quality and yield [61]. Our previous research line employing other local endemic plants of Crete such as V. arcturus [26], O. microphyllum [27], S. syriaca [28] and C. diae [29] offer a first report regarding the evaluation of the total phenolic and flavonoid contents which may further inspire the usage of fertilization in stimulating herbal antioxidant content without compromised optical quality or yield in MAPs. Such investigations are supplementary to the current study dealing with O. dictamnus as they offer insight into several cases of exemplary neglected and underutilized local endemic species of Crete responding in similar ways to fertilization. Furthermore, the fertilization strategies employed in such studies incorporate a multidisciplinary approach dealing with biological, physiological, phytochemical, and agronomical aspects of different species with promising potential in different economic sectors, all being useful for the establishment of distinct species-specific value chains [51].

The fertilization treatments in O. dictamnus affected neither the above-ground plant growth (Figure 5A) nor the tissue water content (Figure 5B). Therefore, the fertilization scheme also did not affect the efficiency of processing (i.e., decrease in moisture content via drying [15,16]). As compared to the control, the fertilization scheme did not affect biomass partitioning to generative organs, besides INM-sa which reduced it (Figure 5C). Among fertilization treatments, plants receiving MPE-sa or INM-sa had lower partitioning to generative organs as compared to the ones receiving ChF-fa (Figure 5C). Considering inflorescences as harvestable organs, the current research suggests that INM-sa treatment should be avoided in O. dictamnus cultivation.

The intensity of greenness of the leaves is a critical parameter of visually perceived quality throughout production, processing, and market outlets [15]. The intensity of leaf green color was here non-invasively determined by employing three methods (SPAD meter, DA meter, Chroma Meter). At the full flowering stage, the fertilization scheme did not affect the leaf SPAD value (Figure 2C), IAD value (Supplementary Figure S1C), a* value (an index of green to red range intensity; Supplementary Figure S3C), and b value (an index of blue to yellow range intensity; Supplementary Figure S4C). An exception to this general trend was the lower L value (an index of lightness; Supplementary Figure S2C) of plants receiving fertilization through the soil (ChF-sa or MPE-sa) as compared to the remaining treatments (Supplementary Figure S2C). When taken altogether, these results indicate that the fertilization scheme did not influence leaf color aspects in O. dictamnus.

Another feature of visual inspection in herbal material grading is leaf outline (shape). The results of the present study denoted that the different fertilization schemes and application methods marginally affected leaf appearance (as this was evaluated herein by the shape indices aspect ratio, roundness, circularity and solidicity). As compared to the control plants, only circularity was changed with MPE-sa (Figure 4B). On this basis and given that color variation was either lacking or limited, it is questionable if instructed staff or computerized identification methods [54] could be employed in visually distinguishing O. dictamnus herbal material that has received fertilization through foliar or soil.

The nutrient contents of leaves and inflorescences (Origani dictamni herba) [43] are largely lacking in the literature regarding O. dictamnus; only a recent pot study was found dealing with the effect of different substrates and sites on nutrient content [45]. Herein, field data have been obtained for the first time, showing variation in the effect of different fertilization schemes depending on the element examined. Thus, regarding the leaf N-P-K content, the ChF-sa, INM-fa and ChF-fa treatments performed well; the same stands for the latter two foliar fertilization schemes in the case of micro-nutrients. Moreover, the INM-fa and the ChF-fa showed the highest values in most cases, indicating the importance of the foliar applications on O. dictamnus nutrition status, especially that of micro-nutrients. Compared to the recently published pot study [45], similar N, P and Zn but lower content values for K were obtained in the present study, while Cu, Fe and Mn were higher in the current study. Regarding the inflorescence nutrient content and specifically concerning N P, K, and Zn, higher values have been reported by a recent study [45] compared to the herein results, showing, however, lower values for Fe. In the latter study, no differences were observed among tested substrates, but the opposite occurred between sites, thus suggesting that environmental conditions might be an important factor. Another finding of the current study was the slight or negligible differences of the inflorescence nutrient content observed between fertilization treatments and the control, indicating a minor effect of fertilization on inflorescence nutrient content. Taken altogether, further work is apparently needed for an extended period to validate the results prior to drawing essential conclusions regarding the accumulation and partitioning of nutrients in plant biomass.

From a phytochemical perspective, medicinal–aromatic plants are generally rich in antioxidant compounds, while their content is an important measure of herbal material quality [17,18]. In this study, three key non-enzymatic antioxidant metabolites (carotenoids, flavonoids, phenols) were determined. As compared to the controls, no negative effect of fertilization on the antioxidant metabolite content was apparent (Figure 6B–D). Considering all three antioxidants together, the INM-fa was the most stimulatory treatment in phytochemical terms, due to the highest (carotenoids, total flavonoids) or the second highest (total phenolics) content as compared to the rest of the treatments (Figure 6B–D). Polysaccharides have been highly connected to antioxidant enhancement using both in vitro chemical and biological models [62]. The polysaccharide antioxidant action is greatly reliant on their solubility, sugar ring assembly, molecular weight, amount of positively or negatively charged clusters, protein moieties and covalently linked phenolic substances [63,64]. The polysaccharide-based INM used in this study proved to be significantly competent in order to enhance the antioxidant machinery in the glandular-haired O. dictamnus. The increased antioxidant machinery, in turn, could be beneficial for both plant resistance to stress (plant health) and product consumer (human health). In accordance with these findings, INM has been previously related to increased antioxidant compound content in other species as well [24].

The second-best stimulatory scheme was ChF-sa, which enhanced the highest (carotenoids) or the second highest (total flavonoids) content as compared to the rest of the treatments (Figure 6B,D). Considering all three antioxidants under study together, the current results indicated that INM-fa should be employed in the commercial cultivation of O. dictamnus. This field investigation presents for the first time, baseline information for some critical herbal quality aspects in O. dictamnus. When inflorescences are the primary target, INM-sa ought to be avoided. When the antioxidant compound content is of key commercial interest, INM-fa stands out as the optimal fertilization scheme in O. dictamnus cultivation.

Soil-applied INM fertilizers are not only absorbed by roots, but foliar-sprayed INM polysaccharides may also be directly absorbed by leaves. Considering all three antioxidants together, INM by foliar application was found to be the optimal scheme. A polysaccharide stock in the plant is vital during the whole growth period, as polysaccharides act as a direct energy source helping plants to cope and survive stress conditions. The positive outcome of INM on the enhancement of the antioxidant mechanism in O. dictamnus could be further ascribed to the activation of various subsequent functions inside plant tissues, such as the elevated hydrolysis of the unique species-specific endogenous subcuticular-located polysaccharides and/or the hydrolysis of the exogenously added polysaccharides provided by INM foliar application. As suggested by other studies, the latter, in turn, may eventually lead to the increase in the contents of physiologically active sugars, thus providing adequate biological resources for the synthesis of complex antioxidants.

5. Conclusions and Prospects

In this field study, the fertilization scheme optimally driving plant growth and herbal material quality was investigated in O. dictamnus, a threatened local endemic of Crete (Greece) of high medicinal value and commercial interest. Different treatments encompassing conventional inorganic fertilizers (foliar/soil application), INM (foliar/soil application), and INM with biostimulant (soil application) were comparatively examined. Beyond its role as a plant nutrient provider, INM is known to feed the soil biota due to its rich polysaccharide content. The data herein provided insights to the polysaccharide-based INM foliar spray system as an EEF system significantly enhancing the antioxidant machinery in O. dictamnus, thus, opening the way towards its commercial use in material sciences as well as in the food and pharmaceutical sectors. The identification of new bioactive compounds, the determination of the vegetation period and the nutrient status during cultivation, the investigation of adulteration, and the quality control of the cultivated O. dictamnus will further unravel new marketing possibilities for the development of evidence-based new phytotherapeutics.