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Article

Mycorrhization of Black Crowberry (Empetrum nigrum L.) After In Vitro Propagation with Mineral Fertilizers

by
Ivan Nechiporenko
1,2,*,
Svetlana Akimova
2,* and
Natalia Semenova
3
1
State Scientific Institution “All-Russian Research Institute of Phytopathology”, St. Institute, Own 5, Bolshie Vyazemy, Odintsovo District, Moscow Region, 143050 Big Vyazyomy, Russia
2
Institute of Horticulture and Landscape Architecture, Russian State Agrarian University—Moscow Timiryazev Agricultural Academy, Timiryazevskaya St., 49, 127434 Moscow, Russia
3
Prokhorov General Physics Institute of the Russian Academy of Sciences, Vavilov Str. 38, 119991 Moscow, Russia
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(9), 1063; https://doi.org/10.3390/horticulturae11091063
Submission received: 31 July 2025 / Revised: 27 August 2025 / Accepted: 2 September 2025 / Published: 4 September 2025
(This article belongs to the Special Issue Innovative Micropropagation of Horticultural and Medicinal Plants)
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Versions Notes

Abstract

Empetrun nigrum L. is a rare berry crop with a high content of biologically active substances, which are of great interest in medicine. In order to obtain sufficient transplants of this crop, in vitro propagation is preferred over other methods. It is known that a large percentage of propagated plants can be lost or damaged, not only at the stage of adaptation to non-sterile conditions, but also in the post-adaptation period. Plants may have weak growth due to poor root development and a lack of nutrients. Therefore, to improve the understanding of the plant requirements in the post-adaptation period of E. nigrum, clonal micropropagation was performed by combining mycorrhizal preparations containing (1) mycelium of Glomus sp., and humic substances, and (2) mycelium of Glomus sp., Trichoderma harzianum, and other mycorrhiza formation microorganisms with different types of mineral fertilizer (N20P20K20, N20P16K10(S5), and N19P9K10 + 2MgO). Analysis of the growth dynamics of ex vitro plants over 98 days of cultivation in containers under greenhouse conditions showed the effectiveness of mineral fertilizer and mycorrhizal preparations treatment. The total root length increased by 30–50% and the total shoot length by 40–80%. The survival was 95.8–100%.

1. Introduction

Crowberry (Empetrum sp.) is a rare berry crop which belongs to the Empetraceae family [1,2,3]. Due to the similarity of morphological characteristics and common habitats with plants such as Erica carnea L. and Phyllodoce empetriformis (Sm.) D. Don., it is sometimes classified as belonging to the Ericaceae family [4,5].
This botanical genus is divided into two large groups of species with different habitats. Plants in the northern hemisphere of Eurasia mainly have black fruits and are grouped under the main species E. nigrum L. Meanwhile, American species have fruits of various colors: pink (E. eamesii Fern. & Wieg.), red (E. rubrum Vahl ex Willd), purple, or violet (E. atropurpureum Fern. & Wieg.) [4].
Black crowberry (E. nigrum L.) is a groundling, creeping, evergreen shrub belonging to a widespread species whose habitat is associated with both forested and treeless mountain and arctic tundra zones [6]. It is highly tolerant of its habitat; however, it reacts extremely negatively to environmental pollution [7]. It is known that in the struggle for competition and resources, crowberry is capable of allelopathic action, suppressing various flora in its habitat [8,9]. It has been found that species of the genus Empetrum release phenolic compounds from their leaves into the soil, causing chemical effects and suppressing other plants. The main phenolic compound responsible for this effect is the allelopathic compound batatasin-III [10]. It is known that crowberry can grow on soils of a wide range of acidity (pH 2.5–7.5), but the crop cannot survive in areas with alkaline soils with a pH of 8.0 and more [11] because of the presence of various mycorrhizal symbiotic fungi on the roots, mainly ericoid mycorrhiza, which die in such conditions [9,12].
Crowberry belongs to a group of wild berry plants classified as “superfoods,” which also includes such plants as bilberry (Vaccinium myrtillus L.), lingonberry (Vaccinium vitis-idaea L.), and cloudberry (Rubus chamaemorus L.), all of which have a high content of biologically active substances. The indigenous peoples of Scandinavia, Russia, and Canada use berries of E. nigrum L. as raw materials for the production of various preparations (jams, pastilles, beverages), but only in the northern regions of natural growth are the fruits a commercial crop [13,14,15,16,17,18]. Crowberries are also used as a natural dye [19]. Since this berry plant is rare in horticulture, there is no information about the existence of its industrial plantations.
In urban areas, crowberry has broad prospects as a medicinal plant raw material containing a large amount of flavonoids, widely used in the pharmaceutical industry [20,21]. Therefore, interest in this berry and medicinal plant is currently growing.
E. nigrum is usually propagated vegetatively by softwood and hardwood cuttings [22]. However, the rate of propagation using these methods is ineffective for reproducing a sufficient amount of plant material. Seed propagation is used in forest nurseries to restore forest ecosystems. However, this method does not preserve the characteristics of the original plant [2], and it is slower than in morphologically similar plants of the Ericaceae family, such as the Calluna and Erica genera [9]. The problem of prolonged seed germination is associated with the hardening of the endocarp, while only a small number of seeds germinate the following spring after stratification under natural conditions [4,23,24].
Thus, there is currently a lack of planting material or black crowberry, and demand for developing accelerated methods of vegetative propagation is increasing. For plants that are difficult to propagate, the most effective technology for multiplication is clonal micropropagation (in vitro), which successfully develops both in industrial propagation and scientific research [25,26,27]. When using this in vitro technology, it is possible to propagate and obtain genetically uniform high-quality planting material in the required quantity [28,29,30].
The key stage in obtaining high-quality planting material after in vitro technology and their further sale in a nursery is the of growing, completion, or post-adaptation of ex vitro plants [31]. There is a lack of information on how plants develop after clonal micropropagation in vitro during ex vitro adaptation and when grown in containers in greenhouse conditions before planting ex vitro plants in the field.
At the same time, there is evidence showing that ex vitro microplants during adaptation in non-sterile conditions may experience increased stress. The latter can cause slowed growth, suppression, and even death of ex vitro plants, ultimately affecting further development during container cultivation. Survival rates after adaptation can vary from 35 to 90%, depending on the species [32,33,34].
One of the reasons for the slow growth of black crowberry when grown in containers may be a lack of nutrition and poor nutrient uptake due to an underdeveloped root system [35]. To eliminate unfavorable factors for better survival and root system stimulation, it is advisable to add special preparations to the substrate which contain various additives capable of stimulating vegetative growth, increasing immunity, for example, agrobacteria, bacterial derivatives (protein preparations), and spores of mycorrhizal fungal [32,36,37,38,39,40,41].
The natural habitat of ericaceous plants is associated with acidic soils and symbiotic relationships with mycorrhizae; thus, preparations containing mycorrhizal fungi may be the most preferable plant cultivation after in vitro conditions [42,43].
These fungi are able to stimulate the formation of new roots and improve the absorption of applied mineral fertilizers due to the fact that the produce dissolves enzymes that convert insoluble minerals into soluble bioavailable forms that are easily used by the plant [44,45,46,47]. They produce extracellular phosphatases and proteolytic enzymes that provide access to difficult-to-decompose organic compounds by decomposing and absorbing the released products. These can then be transferred to the host plant, thereby increasing the supply of nitrogen and phosphorus. This contributes to the potential competitive advantage of ericaceous plants, as they have access to soil organic matter [48].
It is noted that ambient temperature can also affect the absorption of nutrients such as nitrogen [49]. In particular, the carbon/nitrogen ratio shifts due to the warming of carbon being supplied to mycorrhizal fungi. Later, the ratio changes to the opposite. Ultimately, a lower carbon/nitrogen exchange ratio may allow Empetrum sp. plants to redirect carbon to other functions—growth, respiration, etc. [50]. The plants are also known to accumulate trace elements in various parts of plants that are normally inaccessible [51].
Mycorrhizal fungi are divided into various groups. There are ericoid mycorrhizal fungi (ErM), which are found only in acidophilic plants of the Ericaceae family that grow in the wild [52,53].
Ericoid mycorrhizal fungi are associated with the root surface of the host plant, forming a loose network of hyphae. These hyphae colonize the cortical cells of the host plant roots and form characteristic intracellular spirals. Only a few taxonomically diverse species of ericoid mycorrhizal fungi have been identified as ericoid mycorrhizae. Most fungi that form ericoid mycorrhizae remain unidentified because they are sterile. The best-known species is Hymenoscyphus ericae, which is believed to colonize both the Ericaceae and Cupressaceae families [9,54,55,56].
Arbuscular mycorrhizal fungi (AMF) belong to the Glomeromycota family, where the species diversity is limited to 350 species described to date. The most common AM fungi belong to the Glomus genus and form a symbiotic relationship with 80% of land plant species, the growth of which depends on them [57,58]. Rhizophagus irregularis is the main species used to study AM and is a key component of various mycorrhizal plant biostimulants, being widely used in horticulture and plant cultivation [59]. Symbiosis with AM is characterized by their penetration of fungi into root the cells of the root cortex with the formation of microscopic, branched structures called arbuscular structures. These structures increase the efficiency of metabolite exchange between the plant and the fungus. The extraradical hyphae of AM create a surface for colonization by functional populations of bacteria [60].
Several studies have reported on the interactions between nitrogen-fixing fungi and bacteria that mobilize phosphorus and nitrogen, as well as the impact of nitrogen fixation on the structure of bacterial community [61,62]. It has been noted that the artificial infection of young, actively growing seedlings with mycorrhizal fungi improves rooting and early plant growth, as there is a need to increase the above-ground system during this growth period [63,64,65]. However, this cannot be achieved without increasing the absorption capacity of the roots and mineral nutrition improvement [66,67]. This is due to the fact that ericaceous plants do not have root hairs at their root tips and are able to absorb nutrients only through a symbiotic relationship with mycorrhizal fungi [68,69]. Therefore, for better plant development, it is necessary to direct all agricultural techniques to create conditions that stimulate the development of mycorrhizal fungi and optimize the conditions for the development of perennial plantings [70].
In view of the rise in prices of mineral fertilizers, the combined use of mycorrhizal fungi can help to reduce fertilizer consumption and improve its absorption [71,72].
Therefore, the aim of our research was to investigate the effectiveness of adding mycorrhizal preparations and various types of fertilizers (water-soluble and slow-released) using different application methods when growing black crowberry plants in greenhouses after in vitro propagation.

2. Materials and Methods

2.1. In Vitro Culture Conditions

The experiments were carried out threefold in 2024–2025 at the Federal State Budgetary Scientific Establishment the All-Russian Scientific Research Institute of Phytopathology (VNIIF), in the Department of the Laboratory for Recovery and Research on the Adaptive Potential of Cultures and Plants, and at the Russian State Agrarian University, Moscow Timiryazev Agricultural Academy, in the department of biotechnology and berry crops of the Edelstein Educational Scientific and Production Center for Horticulture and Vegetable Growing.
Crowberry microplants were obtained using in vitro propagation. At the stage of initiation of in vitro culture, cut young shoots of E. nigrum L. cv. ‘Irland’ (European variety) were used (Figure 1). They were washed under running water together with a soap solution. Shoots were sterilized in a laminar box using 3% sodium hypochlorite (NaClO) with an addition of 0.01% polysorbate (Tween 80) for 15 min and 70% ethanol for 1 min. Then the shoots were then washed with bidistilled water threefold, cut into 1.0 cm pieces, and planted on Woody Plant Medium (Lloyd & McCown, 1980) [73] with an addition of 0.2 µM 6-BA (6-benzylaminopurine) (Duchefa Biochemie, Haarlem, The Netherlands).
At the stage of multiplication, three sequential passages were produced on the nutritive growth medium based on WPM enriched with the following substances (mg/L): thiamine hydrochloride (B1); pyridoxine hydrochloride (B6); nicotinamide (PP), 0.5; meso-inositol, 100; and sucrose, 30.000, with added cytokinin of Zeatin (2-Methyl-4-(9H-purin-6-ylamino)-2-buten-1-ol) (HiMedia Laboratories LLC, Maharashtra, India) at a concentration of 2.2 µM and 0.8% w/v agar-agar (American-type bacteriological) (Dia-m, Moscow, Russia), with the pH adjusted to 4.5 with 1M NaOH or HCl. The plants in vitro were cultivated in a controlled environment room with a photosynthetic photon flux density (PPFD) of 26.62 µmol·s−1·m−2, a 16/8 h (light/dark) photoperiod, and temperature of 20 ± 2 °C. The subcultivation period was 60 days.
At the rooting stage, in vitro microcuttings were planted on the nutritive growth medium based on ½WPM (reduction of macronutrients and sucrose) with added IAA (indolyl-3-acetic acid) (Duchefa Biochemie, Haarlem, The Netherlands) at a concentration of 12.3 µM. Rooting in vitro plants was subcultivated with a PPFD of 26.62 µmol·s−1·m−2, with a 16/8 h (light/dark) photoperiod and 20 ± 2 °C temperature. The subcultivation period was 60 days.

2.2. Adaptation Conditions

At the adaptation stage, microplants were replaced in industrial chambers—dome-shaped mini-greenhouses with holes (for ventilation) (with temperature conditions of 22 s 2 °C, humidity 90%)—and located under Zěma ZML-0160 LED phytolamps (Ecolight, Moscow, Russia) with a level PPFD of 120 µmol·s−1·m−2 and a 16/8 h (light/dark) photoperiod for 45 days. After adaptation to non-sterile conditions, the plants were kept in a greenhouse box (temperature 22–30 °C, humidity 70–75%).

2.3. Experimental Treatments

In the first ten days of March, the experimental adapted crowberry plants were replaced in 0.5 L containers (square pot 0.5 L 9 × 9 × 10 cm) in high-moor peat substrate (pH ≤ 4.0), to which mycorrhiza-forming preparations containing arbuscular mycorrhizal fungi of the genus Glomus were added according to the experimental design. The mycorrhizal preparations ‘Kormilitsa’ (BashInkom, Ufa, Russia) and ‘Mycofriend’ (Organic Line, Lytkarino, Russia) were used.
In addition, to ensure better plant survival, growth and development mineral fertilizers were applied with mycorrhiza combination and without it (Table 1).
‘Plantafol’ (Valagro®, Chieti, Italy)—chelated N20P20K20 fertilizer with microelements, which has increased solubility and complete absorption—was applied for foliar treatment seven times with 14-day intervals (pH of 1% solution: 4.5).
‘Rastvorin’ (Buyskiy Himicheskiy Zavod, Buy, Russia)—water-soluble fertilizer for heather was used for root nutrition N20P16K10(S5) seven times, once every 14 days (pH of 1% solution—3.0).
‘Osmocote PRO’ 3–4M (ICL Group, Glebe, The Netherlands)—a slow-release fertilizer N19P9K10 + 2MgO with trace elements—was used to enrich the substrate.
Thus, by combining mineral fertilizers and mycorrhizal additives and using them separately, we obtained 12 experimental variants (Table 2).
pH measurements were performed using an Expert-001 pH meter-ionomer (Ekoniks-Expert, Moscow, Russia).
Morphometric indicators of ex vitro plant development were recorded seven times, every two weeks, on the 14th, 28th, 42nd, 56th, 70th, 84th, and 98th days of cultivation. The following parameters were recorded: number (pcs.) and length of shoots (cm), total shoot length (cm), and, at the end of the last data recording, number (pcs.) and length of roots (cm), as well as total root length (cm).

2.4. Data Analysis

The experiments were repeated three times, with eight plants used for each repetition. The statistical data processing was first performed using multi-way analysis of variance (ANOVA), after which significant differences between the mean values for variant were assessed using Duncan’s multiple range test (p ≤ 0.05). This confirmed the reliability of the research results. The data are presented as mean values and the standard error of the mean (M ± SEM).

3. Results

During the ex vitro cultivation of E. nigrum ‘Irland’ plants in a greenhouse, the survival and morphometric indicators of plant development were recorded every 14 days. It was found that the survival of plants during 98 days of cultivation was 100% in the control variant (T1) without fertilizers or mycorrhiza, in variants with the mycorrhiza-forming preparation separately T2 or with N20P16K10(S5) (T9) fertilizer, in variants with the N20P20K20 fertilizer (T4) or with mycorrhiza containing fertilizer (T10), and in variants with the mycorrhiza-forming preparation with the N20P16K10(S5) fertilizer (T12) application (Table 3).
The lowest survival (plants on the 98th day of growth: 29.2–75.0%) were observed in variants with the introduction of the slow-release fertilizer N19P9K10 + 2MgO into the substrate (T5), both separately and in combination with mycorrhiza-forming preparations (T8, T11). The best survival of plants was observed in the variant with the combination of the mycorrhiza-forming preparation T3, T10, T12.
The survival of crowberry plants in the other variants of the experiment on the 98th day was 91.7–95.8% (Table 3).
An analysis of seven counts and observations showed that mycorrhizal preparations and mineral fertilizers have a significant impact on the morphometric parameters of adapted plants.
Significant differences were found when measuring the height of E. nigrum experimental plant shoots on the 14th day of cultivation compared to the control in all variants without mycorrhizae (2.8–3.3 cm compared to 1.6 cm in the control T1). Significant differences were also found in the T11 variant where N19P9K10 + 2MgO fertilizers and mycorrhiza-forming preparation were added to the substrate (3.5 cm compared to 2.5 cm in T1) (Figure 2B).
Taking the total shoot length into account, significant differences were also found with the control in all variants without mycorrhizal preparations (3.5–4.2 cm). Additionally, significant differences were observed in all variants of the experiment with the control without mycorrhizae, except for those with foliar applications using N20P20K20 fertilizer (Figure 2C).
On the 28th day of cultivation, the obtained results did not demonstrate reliable differences with the control of all the studied the experimental variants. This is probably due to the biological characteristics of crowberry plants, which in natural habitat conditions grow mainly on soils with a poor composition of nutrients. Due to the fact that our plants were multiplied using the technology of clonal micropropagation and were planted in substrates enriched with mycorrhizae and nutrients, it can be assumed that on the 28th of cultivation, according to development indicators, they were approximately at the same level. The data are presented in Supplementary Figure S1.
However, it should be noted that on the 28th day of cultivation, the N19P9K10 + 2MgO fertilizer was found to have a detrimental effect on the plants, both when used separately (T5) and when used in combination with the mycorrhiza-forming preparation (T8). The plants’ survival was 79.2% at this point and subsequently declined steadily to 29.2–37.5% by the end of the observation period. The data are presented in Supplementary Figure S1.
Records of the number of shoots taken on the 42nd day of cultivation showed significant differences between variants T8 and T9 (1.8–2.1 pcs). Additionally, in variants with the addition of mycorrhizae to the substrate, there were significant differences observed in both the variants without fertilizers (T3) (2.2 pcs) and in combination with the N20P16K10(S5) fertilizer (T12) (2.0 pcs).
Average shoot height observations showed significant differences from the control for all fertilizer application options in experimental variants without mycorrhizae—T4, T5, and T6 (4.5–5.6 cm). The same was observed in variants T10 and T11 (4.8–5.9 cm compared to 3.6 cm in the control, T1).
Data analysis of the total shoot height (Figure S2) showed significant differences from T1 in all variants with the mycorrhizae application to the substrates T4, T10, T11, and T12 (6.8–8.0 cm). The same was observed in the T6, T9, and T12 variants with the addition of the N20P16K10(S5) fertilizer (6.3–8.0 cm).
On the 56th day of cultivation, it was observed that foliar fertilizer N20P20K20 significantly affected shoot height (6.5 cm compared to 4.2 cm in T1) and total shoot length (10.5 cm compared to 5.9 cm in T1) in variants without mycorrhizae (T4). The N20P16K10(S5) fertilizer had the best effect on total shoot length in T9 and T12 variants with mycorrhiza-forming preparations (10.6–10.6 cm). A significant increase in the parameter was also observed in the T3, T11, and T12 variants. The total shoot length was 10.5–10.6 cm in these variants (Figure S3).
On the 70th day of cultivation, the advantage of the previously identified effective treatment options was preserved (Figure S4). The N20P20K20 fertilizer with preparation mycorrhizae had a significant effect on shoot length in the T4 variant (9.3 cm compared to 6.3 cm in T1) and total shoot length without mycorrhizae in the T4 variant (14.5 cm compared to 9.8 cm in T1). The N20P16K10(S5) fertilizer had the best effect on total shoot length in variants with in the T9 and T12 variants (15.4–15.6 cm).
On the 84th day of cultivation, the addition of N20P20K20 fertilizer to variants without mycorrhizae (T4) had a significant effect on both the number of shoots (2.8 pcs. compared to 1.8 pcs. in T1) and the total shoot length (20.3 cm compared to 11.8 cm in T1). The N20P16K10(S5) fertilizer produced the best results in terms of total shoot length in the T9 and T12 variants treated with mycorrhizae (20.5–20.8 cm compared to 14.8 cm in the control without mycorrhizae).
Significant advantages were found in variants with the addition of the mycorrhiza-forming preparation KM to the substrate, both with T10 and T12 and without T3 fertilizer (19.9–20.9 cm compared to 11.8 cm in T1) (Figure S5).
Morphometric indicators of ex vitro E. nigrum plants on the 98th day of cultivation showed that the advantages of the previously selected variants were preserved. The effectiveness of adding N20P20K20 fertilizer without mycorrhiza-forming preparations (T4) was demonstrated, with 5.1 pcs. shoots compared to 1.8 pcs. in T1 and a total shoot length of 25.8 cm compared to 14.9 cm. At the same time, the highest values for shoot length were in the T7 variant (10.1 cm).
In direct application to substrate, the N20P16K10(S5) fertilizer shows significant advantages in total shoot length. This is evident in both variants, with and without mycorrhizae. In the variant without mycorrhizae (T6), the total shoot length was 20.3 cm, compared to 14.9 cm in T1. In the variant with preparations mycorrhizae (T9 and T12), the total shoot length was 25.4–26.1 cm, compared to 14.9 cm in the control variant.
Significant advantages were also confirmed for the T3 and T12 variants with the addition of the mycorrhiza-forming preparation to the substrate, in terms of total shoot length (25.2 cm compared to 14.9 cm in T1) (Figure 3 and Figure 4).
On the 98th day of cultivation of E. nigrum ‘Irland’ plants, the morphometric indicators of the root system development of the experimental plants were recorded after completing the accounting and observations of the above-ground system.
The results confirmed the effectiveness of the previously selected variants and demonstrated the positive impact of foliar application with N20P20K20 fertilizer in the absence of mycorrhizae (T4). This treatment increased root length to 6.9 cm and total root length to 33.3 cm. N20P16K10(S5) fertilizer positively affected total root length in the T6 variant without mycorrhizae (32.7 cm compared to 24.8 cm in T1). It also had a positive effect on total root length in the T9 and T12 variants with mycorrhizae (37.1–37.2 cm compared to 24.8 cm in the control group T1).
The positive effect of the mycorrhiza-forming agent KM on root length (6.5 cm compared to 5.4 cm in T1) and total root length in the no-fertilizer and mineral fertilizer N20P16K10(S5) conditions in the T3 and T12 variants was also revealed to be 33.8–37.6 cm compared to 24.8 cm in the control (Figure 5 and Figure 6).

4. Discussion

Due to the increase in the price of mineral fertilizer, some plant nurseries are already using the practice of combining mycorrhizal preparations and mineral fertilizers, which allow for more rational and effective use of fertilizers [74,75,76,77]. Therefore, it was important to study the peculiarities of the influence of mycorrhizal preparations on the ex vitro cultivation of E. nigrum plants in containers under greenhouse conditions with optimally selected mineral nutrition.
It is known that E. nigrum has a predominantly taproot-type root system that is later replaced by creeping lateral roots without root hairs, located in the upper 5–10 cm soil layer [5,23]. For the epidermal cells of E. nigrum, for the absorption of water and nutrients, symbiosis with ericoid or arbuscular mycorrhizal fungi is necessary [78,79,80,81].
In habitat, crowberry grows in nutrient-poor areas, which determines the level of ash elements. Additionally, nitrogen, phosphorus, and potassium are present in peat bog organic matter in a form that is difficult to assimilate [82,83]. At the same time, phosphorus is unavailable in acidic soils with a large amount of undecomposed and semi-decomposed plant residues (high-moor peat) as it precipitates together with iron and aluminum [84,85,86].
Symbiosis with mycorrhizal fungi develops better when the soils contain low amounts of soluble phosphorus and nitrogen. Therefore, symbiosis between mycorrhizae and plants is of great importance for the adaptation of E. nigrum plants to conditions of low levels of available phosphorus, sulfur, nitrogen, and micronutrients in the soil. In this case, ammonium nitrogen is considered the most suitable source, since it is more available in acidic soils than the nitrate form. Furthermore, it was found that ammonium nitrogen allows for better vegetative growth and root system development in E. nigrum [50,87].
AM is widely used for introduction into the substrate when growing plants of the genus Vaccinium L., since it is capable of increasing adaptive capacity and may have powerful stress-protective properties capable of stimulating development on the root and, subsequently, above-ground parts of experimental plants [88]. This is probably associated with the effectiveness of the experimental treatment options with mycorrhiza-forming preparations of MF and KM, which contain fungi of the genus Glomus.
The high stress-protective and stimulating effect of the mycorrhiza-forming preparation (T3), containing mycelium and spores of fungi of the genus Glomus, on the survival, safety, and development of experimental ex vitro E. nigrum should also be noted. Therefore, during all the recorded observations of the variants, with the inclusion of the mycorrhizal preparations in the substrate composition, both independently and in combination with the fertilizers N19P9K10 + 2MgO and N20P16K10(S5), reliable differences were revealed in all the considered indicators.
The humic substances included in the mycorrhizal-forming preparation (T3) are characterized by a high capacity of cation and anion exchange, the ability to interact with soil enzymes, and vitamins. These activate plant immunity and development [89].
New research shows that not only mycorrhizal fungi, but also some root-symbiotic bacteria, are capable of stimulating ericaceous plants to become resistant to unfavorable environmental conditions [90]. Trichoderma harzianum, which is part of the ‘Mycofriend’ preparation, is a well-known biological agent that occurs in nature and is an endophytic symbiont. Similarly, the genus Bacillus is considered useful in agriculture due to its high level of antagonism towards various phytopathogenic microorganisms [91,92,93,94,95]. Pseudomonas fluorescens is a Gram-negative bacterium with antibacterial properties that has attracted the attention of researchers as an alternative to chemical bactericide agents [37,96,97]. This is probably related to the high survival of crowberry plants in variants using the mycorrhiza-forming preparation (T2, T7, T9).
In order for a plant to grow and develop well, it is necessary to create conditions that stimulate the development of mycorrhizal fungi and optimize the absorption of nutrients by plants. Therefore, in our research, we studied combinations of mycorrhizal preparations with different types of fertilizer (water-soluble and slow-release) with various methods of application (substrate application, direct application to the substrate, and foliar application).
It is known that the slow-release fertilizers N19P9K10 + 2MgO are included in the group of fertilizers with a coating based on organic polymers—they have a multi-layer, porous coating-based alkyd resins. When entering the soil, moisture, under the action of osmotic pressure, penetrates through the pores into the granule, as a result of which it stretches, and through the expanded pores, nutrients enter in their dissolved form [98,99].
It is known that the nutrients of slow-release fertilizers are unaffected by soil salt content, pH level, microbial activity, or water quality. The only important factor is a temperature above +21 °C; above this, the release of nutrients is faster, and below it, it is slower.
Many studies conducted in recent years have shown that acidic or alkaline environments have virtually no effect on the release of nutrients from the polymer coatings of slow-release fertilizers [100,101]. However, our studies revealed no advantages of the slow-release fertilizer N19P9K10 + 2MgO for the ex vitro survival of E. nigrum plants, which had a survival of between 29.2% and 75.0% on the 98th day of cultivation.
We assume that due to the release of phenolic substances by crowberry roots, in particular Batatasin-III, there could be an accelerated release of nutrients contained in the fertilizer granules. For this reason, young roots could get burned, and the plants could die. It is known that phenolic compounds can significantly slow down the rate of nutrient decomposition, impact the ability to accumulate organic matter, and also change the circulation of nitrogen and suppress the saprophytic activity of the soil [102,103].
Nevertheless, other researchers have demonstrated that the pH of the environment in which the release occurs significantly affects the release of nutrients from slow-release fertilizers, particularly when pH-sensitive polymers are used as the coating material [104]. Furthermore, some polymers may undergo structural changes or changes in properties in response to pH changes [105,106].
Depending on the nature of the functional group (e.g., a carboxyl or amino group) and its state (protonation or deprotonation in response to pH changes), electrostatic repulsion between chains may occur. This can lead to expansion or contraction of the chains, which affects polymer swelling. This can potentially either promote or inhibit moisture penetration and nutrient dissolution [102,107].
For example, studies on the release of nitrogen from urea coated with a bio-nanocomposite hydrogel in solutions with different pH levels have shown that nitrogen release increases as the pH rises. Within 24 h, 20.0%, 25.6%, and 34.9% of nitrogen was released in solutions with pH levels of 4.0, 7.0, and 10.0, respectively [108].
Foliar application with N20P20K20 leaf fertilizer containing microelements in a chelated form, which increases solubility and absorption, proved effective without the need for mycorrhiza-forming preparations. When used on the 98th day of cultivation, plant survival was 100%, total root length increased by 1.3 times compared to the control, and the total shoot length increased by 1.7 times. This is probably because foliar application results in the rapid absorption of nutrients due to the fertilizer interacting most effectively with the physiological processes of the foliage [109,110].
The best results were obtained by direct application to substrate-experimental E. nigrum plants with a water-soluble fertilizer for heathers containing sulfur N20P16K10(S5), both separately and in combination with mycorrhiza-forming preparations. The survival on the 98th day of cultivation was 95.8–100%, and the total root and shoot length increased by 1.4–1.5 times and 1.4–1.8 times, respectively, compared to the control.
In forest and berry plant nurseries, sulfur fertilizers are used to acidify the soil [111,112]. The importance of sulfur (S) is due to the fact that it is a component of protein and non-protein amino acids, it plays a crucial structural role in cells in the form of disulfide bonds in proteins, and it participates in enzyme regulation and provides protection against oxidative stress with the help of glutathione. However, plant species vary greatly in their sulfur requirements. The assimilation of sulfur and nitrogen is closely interrelated [113]. Sulfur also plays an important role in protecting plants against disease [111,114]. Various plant species prevent fungal infections by depositing elemental sulfur in the xylem parenchyma [115].
However, sulfur deficiency can lead to reduced root development and a decrease or change in root exudates, affecting the community of microorganisms that use them as a carbon source [116,117,118]. Plant root activity affects the physical and chemical properties of soil by releasing organic compounds (rhizodeposition), accounting for 15–30% of photosynthetically produced carbon. This process provides soil organisms with energy to perform their functions. Thus, there is a synergistic effect between mycorrhizal preparations and sulfur-containing fertilizers.

5. Conclusions

The results obtained have improved our understanding of the conditions for ex vitro cultivation black crowberry of the ‘Irland’ variety using various mycorrhiza-forming preparations and different types of fertilizers.
On the 98th day of cultivation, the advantage of adding the mycorrhizal preparation granular mycorrhizae containing only mycelium of the genus Glomus to the substrate, both separately and with the mineral fertilizer N20P16K10(S5), was revealed (the total root length of the plant increased by 43–50%, and the total shoot length increased by 70%). Also, when using the mycorrhizal preparation KM, the survival on the 98th day of cultivation was 95.8%, and when combined with the water-soluble fertilizer N20P16K10(S5), it was 100%. In addition, it was found that it is effective to apply N20P16K10(S5) fertilizer every 14 days, both independently and in combination with mycorrhiza-forming preparations (increase in total root length by 31–51% and total shoot length by 43–88%).
Without mycorrhizal preparations, it is effective to use foliar applications of the fertilizer N20P20K20 every 14 days, which increases the total root length by 34% and the total shoot length by 72%, at the same time, and the survival on the 98th day of cultivation is 100%.
At the same time, the worst variants were found where the slow-release fertilizer N19P9K10 + 2MgO was applied in the substrate. In T5 and T8 variants, the survival on the 98th day of cultivation of experimental plants was 29.2–37.5%.
This study highlights the importance of using mycorrhizal preparations in combination with different types of fertilizers during the post-adaptation period of E. nigrum plants to obtain more vigorous and stress-resistant plants for cultivation in plant nurseries, and then in industrial plantations for fruit.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11091063/s1, Figure S1. Morphometric indicators (A—number of shoots, B—shoot height, C—total shoot length) of ex vitro development of E. nigrum ‘Irland’ plants on the 28th day of cultivation. Figure S2. Morphometric indicators (A—number of shoots, B—shoot height, C—total shoot length) of ex vitro development of E. nigrum ‘Irland’ plants on the 42nd day of cultivation. Figure S3. Morphometric indicators (A—number of shoots, B—shoot height, C—total shoot length) of ex vitro development of E. nigrum ‘Irland’ plants on the 56th day of cultivation. Figure S4. Morphometric indicators (A—number of shoots, B—shoot height, C—total shoot length) of ex vitro development of E. nigrum ‘Irland’ plants on the 70th day of cultivation. Figure S5. Morphometric indicators (A—number of shoots, B—shoot height, C—total shoot length) of ex vitro development of E. nigrum ‘Irland’ plants on the 84th day of cultivation.

Author Contributions

Conceptualization, I.N.; methodology, I.N. and S.A.; formal analysis, I.N. and N.S.; investigation, I.N. and S.A.; resources, I.N.; data curation, I.N., S.A. and N.S.; writing—original draft preparation, I.N.; writing—review and editing, I.N. and S.A.; visualization, I.N.; supervision, S.A.; project administration, S.A. and N.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Data is contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Horticulturae 11 01063 g001
Figure 1. The process of obtaining adapted ex vitro plants of E. nigrum cv. ‘Irland’: (A) explant introduced into in vitro culture; (B) in vitro microcuttings at the multiplication stage; (C) microcuttings at the in vitro rooting stage; (D) ex vitro microplants in the adaptation process; (E) adapted ex vitro microplant with roots; (F) ex vitro plants in the process of growing in a greenhouse.
Figure 1. The process of obtaining adapted ex vitro plants of E. nigrum cv. ‘Irland’: (A) explant introduced into in vitro culture; (B) in vitro microcuttings at the multiplication stage; (C) microcuttings at the in vitro rooting stage; (D) ex vitro microplants in the adaptation process; (E) adapted ex vitro microplant with roots; (F) ex vitro plants in the process of growing in a greenhouse.
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Figure 2. Morphometric indicators ((A) number of shoots, (B) shoot height, (C) total shoot length) of ex vitro development of E. nigrum ‘Irland’ plants on the 14th day of cultivation. Values represent mean SEM (n = 8). Letters indicate significant differences among treatments according to Duncan’s test (p ≤ 0.05).
Figure 2. Morphometric indicators ((A) number of shoots, (B) shoot height, (C) total shoot length) of ex vitro development of E. nigrum ‘Irland’ plants on the 14th day of cultivation. Values represent mean SEM (n = 8). Letters indicate significant differences among treatments according to Duncan’s test (p ≤ 0.05).
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Figure 3. Morphometric indicators ((A) number of shoots, (B) shoot height, (C) total shoot length) of ex vitro development of E. nigrum ‘Irland’ plants on the 98th day of cultivation. Values represent mean SEM (n = 8). Letters indicate significant differences among treatments according to Duncan’s test (p ≤ 0.05).
Figure 3. Morphometric indicators ((A) number of shoots, (B) shoot height, (C) total shoot length) of ex vitro development of E. nigrum ‘Irland’ plants on the 98th day of cultivation. Values represent mean SEM (n = 8). Letters indicate significant differences among treatments according to Duncan’s test (p ≤ 0.05).
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Figure 4. The dynamics of changes in the total shoot length of experimental E. nigrum ‘Irland’ plants, depending on the addition of mycorrhizal preparations and mineral fertilizers application.
Figure 4. The dynamics of changes in the total shoot length of experimental E. nigrum ‘Irland’ plants, depending on the addition of mycorrhizal preparations and mineral fertilizers application.
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Figure 5. Morphometric indicators ((A) number of roots, (B) root height, (C) total root length) of E. nigrum ‘Irland’ plants on the 98th day of cultivation. Values represent mean SEM (n = 8). Letters indicate significant differences among treatments according to Duncan’s test (p ≤ 0.05).
Figure 5. Morphometric indicators ((A) number of roots, (B) root height, (C) total root length) of E. nigrum ‘Irland’ plants on the 98th day of cultivation. Values represent mean SEM (n = 8). Letters indicate significant differences among treatments according to Duncan’s test (p ≤ 0.05).
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Figure 6. The effect of adding mycorrhiza-forming preparations and mineral fertilizers to the substrate on the morphometric indicators of the development of experimental E. nigrum ‘Irland’ plants on the 98th day of cultivation.
Figure 6. The effect of adding mycorrhiza-forming preparations and mineral fertilizers to the substrate on the morphometric indicators of the development of experimental E. nigrum ‘Irland’ plants on the 98th day of cultivation.
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Table 1. Composition, method of application, and concentration of mycorrhiza preparations and mineral fertilizers.
Table 1. Composition, method of application, and concentration of mycorrhiza preparations and mineral fertilizers.
Mycorrhiza and FertilizersCompositionApplication MethodConcentration
(g/L)
Mycorrhiza forming additives
KormilitsaGranular mycorrhizae containing mycelium and spores of fungi of the genus Glomus, colonized root fragments, natural organic fillers, humic substancesSubstrate application6
MycofriendPowdered preparation containing
fungi (Glomus sp., Trichoderma harzianum);
microorganisms that support the formation of mycorrhizae and plant rhizosphere (Streptomyces sp., Pseudomonas fluorescens);
phosphate-solubilizing bacteria with fungicidal and bactericidal properties (Bacillius megaterium var. phosphaticum, Bacillus subtilis, Bacillus muciloginosus, Enterobacter sp.);
total number of viable cells (1.0–1.5) × 108 CFU/g;
biologically active substances (phytohormones, vitamins, amino acids)		Substrate application0.4
Mineral fertilizers
PlantafolN—20% (N-NO3—4%, N-NH4—2%, N-NH2—14%), P2O5—20%, K2O—20%, microelement chelates with EDTA (B-0.02%; Fe-0.01%; Mn-0.05%; Cu-0.005)Foliar application2
RastvorinN—20%, P2O5—16%, K2O—10%, S-5.5%, Microelements: B-0.01%; Mn-0.1%; Cu-0.01%; Zn-0.01%; Mo-0.001%Direct application to substrate2
Osmocote PRON—19% (N-NO3—6.6%, N-NH4—9%, N-NH2—1.4%), P2O5—9%, K2O—10%, MgO-2%, Microelements: B-0.01%; Fe-0.007%; Mn-0.04%; Cu-0.023%; Zn-0.011%; Mo-0.01%, Substrate application2
Table 2. Experimental treatment variants.
Table 2. Experimental treatment variants.
Treatment VariantMycorrhizaMineral Fertilizers
KormilitsaMycofriendPlantafolRastvorinOsmocote PRO
T1
T2 +
T3+
T4 +
T5 +
T6 +
T7 ++
T8 + +
T9 + +
T10+ +
T11+ +
T12+ +
‘+’ means that it is used in this treatment variant.
Table 3. Survival of E. nigrum ‘Irland’ plants ex vitro during greenhouse cultivation in containers. Values represent mean SEM (n = 8). Letters indicate significant differences among treatments, ns—indicates non-significant differences according to Duncan’s test (p ≤ 0.05).
Table 3. Survival of E. nigrum ‘Irland’ plants ex vitro during greenhouse cultivation in containers. Values represent mean SEM (n = 8). Letters indicate significant differences among treatments, ns—indicates non-significant differences according to Duncan’s test (p ≤ 0.05).
VariantPlant Survival, %
14th Day28th Day42nd Day56th Day70th Day84th Day98th Day
T1100.0 ± 0 ns100.0 ± 0 c100.0 ± 0 d100.0 ± 0 e100.0 ± 0 e100.0 ± 0 e100.0 ± 0 e
T2100.0 ± 0 ns100.0 ± 0 c100.0 ± 0 d100.0 ± 0 e100.0 ± 0 e100.0 ± 0 e100.0 ± 0 e
T3100.0 ± 0 ns95.8 ± 4.2 b95.8 ± 4.2 cd95.8 ± 4.2 de95.8 ± 4.2 de95.8 ± 4.2 de95.8 ± 4.2 de
T4100.0 ± 0 ns100.0 ± 0 c100.0 ± 0 d100.0 ± 0 e100.0 ± 0 e100.0 ± 0 e100.0 ± 0 e
T5100.0 ± 0 ns79.2 ± 8.5 a58.3 ± 10.3 b45.8 ± 10.4 b41.6 ± 10.1 b37.5 ± 10.1 b37.5 ± 10.1 b
T6100.0 ± 0 ns95.8 ± 4.2 b95.8 ± 4.2 cd95.8 ± 4.2 de95.8 ± 4.2 de95.8 ± 4.2 de95.8 ± 4.2 de
T7100.0 ± 0 ns95.8 ± 4.2 b91.7 ± 5.8 c91.7 ± 5.8 d91.7 ± 5.8 d91.7 ± 5.8 d91.7 ± 5.8 d
T8100.0 ± 0 ns79.2 ± 8.5 a37.5 ± 10.1 a37.5 ± 10.1 a33.3 ± 9.8 a29.2 ± 9.5 a29.2 ± 9.5 a
T9100.0 ± 0 ns100.0 ± 0 c100.0 ± 0 d100.0 ± 0 e100.0 ± 0 e100.0 ± 0 e100.0 ± 0 e
T10100.0 ± 0 ns100.0 ± 0 c100.0 ± 0 d100.0 ± 0 e100.0 ± 0 e100.0 ± 0 e100.0 ± 0 e
T11100.0 ± 0 ns95.8 ± 4.2 b95.8 ± 4.2 cd83.3 ± 7.8 c83.3 ± 7.8 c75.0 ± 9.0 c75.0 ± 9.0 c
T12100.0 ± 0 ns100.0 ± 0 c100.0 ± 0 d100.0 ± 0 e100.0 ± 0 e100.0 ± 0 e100.0 ± 0 e
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Nechiporenko, I.; Akimova, S.; Semenova, N. Mycorrhization of Black Crowberry (Empetrum nigrum L.) After In Vitro Propagation with Mineral Fertilizers. Horticulturae 2025, 11, 1063. https://doi.org/10.3390/horticulturae11091063

AMA Style

Nechiporenko I, Akimova S, Semenova N. Mycorrhization of Black Crowberry (Empetrum nigrum L.) After In Vitro Propagation with Mineral Fertilizers. Horticulturae. 2025; 11(9):1063. https://doi.org/10.3390/horticulturae11091063

Chicago/Turabian Style

Nechiporenko, Ivan, Svetlana Akimova, and Natalia Semenova. 2025. "Mycorrhization of Black Crowberry (Empetrum nigrum L.) After In Vitro Propagation with Mineral Fertilizers" Horticulturae 11, no. 9: 1063. https://doi.org/10.3390/horticulturae11091063

APA Style

Nechiporenko, I., Akimova, S., & Semenova, N. (2025). Mycorrhization of Black Crowberry (Empetrum nigrum L.) After In Vitro Propagation with Mineral Fertilizers. Horticulturae, 11(9), 1063. https://doi.org/10.3390/horticulturae11091063

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