The effect of seasonal changes in mineral nutrition and secondary metabolism of Epipactis palustris (Orchidaceae) on the rhizosphere soil ecosystem

N. V. Zaimenko; N. S. Chernikova; N. P. Didyk; N. A. Pavliuchenko; O. P. Yunosheva; I. P. Kharytonova; O. V. Zakrasov; A. M. Gnatiuk; M. B. Gароnenko

doi:10.15421/0226074

N. V. Zaimenko M. M. Gryshko National Botanical Garden оf National Academy of Sciences of Ukraine
N. S. Chernikova M. M. Gryshko National Botanical Garden оf National Academy of Sciences of Ukraine
N. P. Didyk M. M. Gryshko National Botanical Garden оf National Academy of Sciences of Ukraine
N. A. Pavliuchenko M. M. Gryshko National Botanical Garden оf National Academy of Sciences of Ukraine
O. P. Yunosheva M. M. Gryshko National Botanical Garden оf National Academy of Sciences of Ukraine
I. P. Kharytonova M. M. Gryshko National Botanical Garden оf National Academy of Sciences of Ukraine
O. V. Zakrasov M. M. Gryshko National Botanical Garden оf National Academy of Sciences of Ukraine
A. M. Gnatiuk M. M. Gryshko National Botanical Garden оf National Academy of Sciences of Ukraine
M. B. Gароnenko M. M. Gryshko National Botanical Garden оf National Academy of Sciences of Ukraine

Keywords: rare plants, nutrient cycling, allelopathic activity, brassinosteroids, soil microbiome, plant adaptation.

Abstract

Epipactis palustris (L.) Crantz is a rare orchid species whose ecological and biochemical strategies of adaptation remain insufficiently studied, despite its relevance for biodiversity conservation and ecosystem stability. This research aimed to investigate the seasonal changes in mineral nutrition, secondary metabolism, enzymatic activity, allelopathic potential, and microbial composition of the rhizosphere soil. Fieldwork was conducted in 2024 at the M. M. G ryshko National Botanical Garden of the National Academy of Sciences of Ukraine (Куiv). The study revealed that E. palustris demonstrates pronounced physiological and biochemical flexibility during the growing season, maintaining mineral balance even under conditions of reduced soil macro- and microelement availability. Elevated accumulation of Ca, Mg, Sr, and Si in the leaves indicated selective ion uptake and the development of ion-homeostatic mechanisms, which are crucial for structural stability and seasonal adaptation. Stable levels of brassinosteroids in the plants throughout the season reflected its ability to sustain hormonal–mineral regulation of growth, photosynthetic activity, and antioxidant defense. In summer, decreased laccase activity and humus content, combined with increased bicarbonate concentration and soil electrical conductivity, pointed to intensified transformation of organic matter and mobilization of mineral elements, creating favorable conditions for plant nutrition. The rhizosphere soil exhibited moderate allelopathic activity, supporting symbiotic interactions with microbial communities. Seasonal shifts in microbial complexes suggested dynamic pathways of organic matter turnover, while the consistent presence of nitrogen-fixing microorganisms ensured nitrogen supply. Taken together, these findings highlight the integrated role of mineral nutrition, hormonal regulation, and rhizosphere interactions in maintaining functional stability of E. palustris . The study provides new insights into the mechanisms of adaptation of rare orchids to environmental stress and emphasizes their connection with mycorrhizal associations. Beyond its ecological significance, this work contributes to the development of scientific approaches for in situ and ex situ conservation of rare orchid species, offering a framework for sustainable management of vulnerable plant populations under changing environmental cond i tions.

References

Alnusairi, G. S., Zaid, A., Kour, H., Alharbi, K., & Soliman, M. H. (2023). Ecophysiological and morphological adaptations of plants under temperature stress: Influence of рhytohormones. In: Tripathi, S., Bhadouria, R., Srivastava, P., Singh, R., & Sanayaima Devi, R. (Eds.). Ecophysiology of tropical plants: Recent trends and future perspectives. CRC Press, Boca Raton. Рp. 128–137.

Bao, L., Liu, Y., Ding, Y., Shang, J., Wei, Y., Tan, Y., & Zi, F. (2022). Interactions between phenolic acids and microorganisms in rhizospheric soil from continuous cropping of Panax notoginseng. Frontiers in Microbiology, 13, 791603.

Brantjes, N. B. M. (1981). Ant, bee and fly pollination in Epipactis palustris (L.) Crantz (Orchidaceae). Acta Botanica Neerlandica, 30, 59–68.

Brzosko, E., Bajguz, A., Burzyńska, J., & Chmur, M. (2021). Nectar chemistry or flower morphology – what is more important for the reproductive success of generalist orchid Epipactis palustris in natural and anthropogenic populations? International Journal of Molecular Sciences, 22(22), 12164.

Chechui, O. F. (2021). Biokhimiya roslyn [Plant biochemistry]. V. V. Dokuchaiev Kharkiv National Agrarian University Press, Kharkiv (in Ukrainian).

Dashbaldan, S., Rogowska, A., Pączkowski, C., & Szakiel, A. (2021). Distribution of triterpenoids and steroids in developing rugosa rose (Rosa rugosa Thunb.) accessory fruit. Molecules, 26(17), 5158.

Didukh, Y. P. (Ed.). (2009). Chervona knyha Ukrayiny. Tsarstvo roslyn [Red data book of Ukraine. Plants]. Globalconsulting, Kyiv (іn Ukrainian).

Didyk, N. & Zaimenko, N. (2025). Prospects for allelochemicals utilization for managing the plant-soil-microbiome system in phytostimulation technologies. Agrobiodiversity for Improving Nutrition, Health and Life Quality, 9(2), 228–245.

Djordjević, V., Aćić, S., Kabaš, E., Lazarević, P., Tsiftsis, S., & Lakušić, D. (2022).The orchids of wetland vegetation in the Central Balkans. Diversity, 15(1), 26.

Gałka, M., & Kasper, P. (2011). Palaeobotanical peat analysis on contemporary Epipactis palustris (L.) Crontz. (Marsh Helleborine) sites in northeastern part of Poland. Studia Limnologicaet Telmatologica, 5, 67–75.

Gaponenko, M. (2013). Osoblyvosti introduktsiyi vydiv rodu Eрiрaсtis Zinn (Orchidaceae Juss.) u Natsionalnomu botanichnomu sadu im. M. M. Hryshka NAN Ukrainy [Features of introduction of species of genus Epipactis Zinn (Orchidaceae Juss.) in M. M. Hryshko National Botanical Gardens of the NAS of Ukraine]. Plant Introduction, 57, 27–32 (іn Ukrainian).

Gnatiuk, A. M., Gaponenko, M. B., Honchar, H. Y., & Gaponenko, A. M. (2023). Diversity of Epipactis palustris (L.) Crantz (Orchidaceae) pollinators and visitors in conditions of Kyiv City (Ukraine). Hacquetia, 22(2), 247–262.

Gołaszewska, E., Gadziszewska, J., & Latałowa, M. (2019). First record of orchid subfossil seeds – the abundant occurrence of Epipactis palustris (L.) Crantz and Dactylorhiza spp. seeds in early Holocene sediments from Central Europe. Review of Palaeobotany and Palynology, 265, 1–12.

Greger, M., Landberg, T., & Vaculík, M. (2018). Silicon influences soil availability and accumulation of mineral nutrients in various plant species. Plants, 7(2), 41.

Hättenschwiler, S., & Vitousek, P. M. (2000). The role of polyphenols in terrestrial ecosystem nutrient cycling. Trends in Ecology and Evolution, 15(6), 238–243.

Hawkesford, M. J., Horst, W. J., Kichey, T., Lambers, H., Schjoerring, J., Møller, I. S., & White, P. J. (2023). Functions of macronutrients. In: Rengel, Z., Cakmak, I., & White, P. J. (Ed.). Marschner's mineral nutrition of plants. Fourth edition. Academic Press. Рp. 201–281.

Hu, L., Robert, C. A., Cadot, S., Zhang, X. I., Ye, M., Li, B., Manzo, D., Chervet, N., Steinger, T., van der Heijden, M. G. A., Schlaeppi, K., & Erb, M. (2018). Root exudate metabolites drive plant-soil feedbacks on growth and defense by shaping the rhizosphere microbiota. Nature Communications, 9(1), 2738.

Hürkan, K., Yüksel, M. B., Hürkan, Y. K., & Demir, N. (2019). Determination of total phenolic and flavonoid contents, antioxidant and antimicrobial activities of some important salep orchids. Eskişehir Technical University Journal of Science and Technology C – Life Sciences and Biotechnology, 8(2), 191–202.

Ionița, O., & Jardan, N. (2024). In-situ conservation of Epipactis palustris (L.) Crantz in the Republic of Moldova. Journal of Plant Development, 31(1), 137–145.

Jacquemyn, H., Brys, R., & Hutchings, M. J. (2014). Biological flora of the British Isles: Epipactis palustris. Journal of Ecology, 102(5), 1341–1355.

Jacquemyn, H., Waud, M., Lievens, B., & Brys, R. (2016). Differences in mycorrhizal communities between Epipactis palustris, E. helleborine and its presumed sister species E. neerlandica. Annals of Botany, 118(1), 105–114.

Jin, Z., Gao, L., Zhang, L., Liu, T., Yu, F., Zhang, Z., Guo Q., & Wang, B. (2017). Antimicrobial activity of saponins produced by two novel endophytic fungi from Panax notoginseng. Natural Product Research, 31(22), 2700–2703.

Karthika, K. S., Rashmi, I., & Parvathi, M. S. (2018). Biological functions, uptake and transport of essential nutrients in relation to plant growth. In: Pandey, N. (Ed.). Plant nutrients and abiotic stress tolerance. Springer, Singapore. Рp. 1–49.

Kirkby, E. A. (2023). Introduction, definition, and classification of nutrients. In: Rengel, Z., Cakmak, I., & White, P. J. (Ed.). Marschner's mineral nutrition of plants. Fourth edition. Academic Press. Рp. 3–9.

Kluza-Wieloch, M., & Maciejewska-Rutkowska, I. (2015). Population of Epipactis palustris (L.) Crantz (Orchidaceae) in south-western part of Poznań. Steciana, 19(4), 231–238.

Kluza-Wieloch, M., & Wyrzykiewicz-Raszewska, M. (2016). Current status of the population of Epipactis palustris (L.) Crantz (Orchidaceae) in the Wielkopolska National Park. Steciana, 20(4), 201–207.

Kluza-Wieloch, M., Wyrzykiewicz-Raszewska, M., Drapikowska, M., Celka, Z., & Maciejewska-Rutkowska, I. (2017). Differentiation of Epipactis рalustris (L.) Crantz (Orchidaceae) in habitats altered by man based on species populations within Poznań City (Poland). Pakistan Journal of Botany, 49(1), 207–214.

Kravets, V. S., Kretinin, S. V., Derevyanchuk, M. V., Drach, S. V., Litvinovskaya, R. P., & Khripach, V. A. (2011). Vlijanie nizkikh temperature na uroven’ endogennykh brassinosteroidov [Effect of low temperatures on the level of endogenous brassinosteroids]. Reports of the National Academy of Sciences of Ukraine, 8, 155–159 (in Russian).

Kulkarni, G., Busset, N., Molinaro, A., Gargani, D., Chaintreuil, C., Silipo, A., Giraud, E., & Newman, D. K. (2015). Specific hopanoid classes differentially affect free-living and symbiotic states of Bradyrhizobium diazoefficiens. mBio, 6(5), e01251-15.

Leten, M., Van den Bussche, W., Provoost, S., Bollengier, B., & Meeus, S. (2022). The orchid flora of the Westhoek Nature Reserve (Belgium, De Panne) and the Flemish dunes revisited…. Liparis, 28(2), 1–223.

Lewis, L., & Mielcarek, R. (2021). Observations on named colour variants of Epipactis palustris. Berichteaus den Arbeitskreisen Heimische Orchideen, 38 (2), 49–69.

Maalouf, R., Maalouf, M., & Véla, E. (2024). Epipactis palustris in the Eastern Mediterranean (Lebanon), a surprising peripheral micropopulation challenges us with its conservation. Flora Mediterranea, 34, 127–136.

Mathé, H. (2015). Les Epipactis du nord-est de la France. Bulletin de la Société Française d’Orchidophilie de Lorraine-Alsace, 3-17 (in French).

Melnychuk, M. D., Likhanov, A. F., Kovalenko, T. M., & Kliuvadenko, A. A. (2022). Vtorynni metabolity ta yikh rol’ u systemakh adaptatsiyi i zakhystu roslyn [Secondary metabolites and their role in plant adaptation and defense systems]. Druk, Vinnytsia (in Ukrainian).

Natta, S., Mondol, M. S. A., Pal, K., Mandal, S., Sahana, N., Pal, R., Pandit, G. K., Alam, B. K., Das, S. S., Biswas, S. S., & Kalaivanan, N. S. (2022). Chemical composition, antioxidant activity and bioactive constituents of six native endangered medicinal orchid species from North-Eastern Himalayan region of India. South African Journal of Botany, 150, 248–259.

Pacsai, B., Sisák, I., & Bódis, J. (2022). Epipactis species and their habitat preferences in the western part of Hungary. Acta Botanica Hungarica, 64(3–4), 391–404.

Pandey, N. (2018). Role of plant nutrients in plant growth and physiology. In: Hasanuzzaman, M., Fujita, M., Oku, H., Nahar, K., & Hawrylak-Nowak, B. (Eds.). Plant nutrients and abiotic stress tolerance. Springer, Singapore. Рp. 51–93.

Pavlovic, J., Kostic, L., Bosnic, P., Kirkby, E. A., & Nikolic, M. (2021). Interactions of silicon with essential and beneficial elements in plants. Frontiers in Plant Science, 12, 697592.

Ricci, J. N., Coleman, M. L., Welander, P. V., Sessions, A. L., Summons, R. E., Spear, J. R., & Newman, D. K. (2014). Diverse capacity for 2-methylhopanoid production correlates with a specific ecological niche. The ISME Journal, 8(3), 675–684.

Scavo, A., Abbate, C., & Mauromicale, G. (2019). Plant allelochemicals: Agronomic, nutritional and ecological relevance in the soil system. Plant and Soil, 442(1), 23–48.

Schmidt, M. A., Kreinberg, A. J., Gonzalez, J. M., Halvorson, J. J., French, E., Bollmann, A., & Hagerman, A. E. (2013). Soil microbial communities respond differently to three chemically defined polyphenols. Plant Physiology and Biochemistry, 72, 190–197.

Sharma, N., Chaudhary, A., Sharma, N., Shukla, S., Joshi, N. C., Vishwakarma, K., Singh, P. K., & Mishra, A. (2025). Plant nutrient requirements and nutrient homeostasis in plants. Іn: Joshi, N. C., Leustek, T., & Singh, P. K. (Eds.). Soil health and nutrition management. CAB International, India. Pp. 1–20.

Tonga, J. L., Kamdem, M. H. K., Pagna, J. I. M., Fonkui, T. Y., Tata, C. M., Fotsing, M. C. D., Nkengfack, E. A., Mmutlane, E. M., & Ndinteh, D. T. (2022). Antibacterial activity of flavonoids and triterpenoids isolated from the stem bark and sap of Staudtia kamerunensis Warb. (Myristicaceae). Arabian Journal of Chemistry, 15(10), 104150.

Tripathi, R., Dubey, R. K., Misra, P., Chaturvedi, K. K., Singh, R. P., Nath, P., & Tuteja, N. (2022). Plant mineral nutrition and disease resistance: A significant linkage for sustainable crop protection. Frontiers in Plant Science, 13, 883970.

Vyshnevskyi, V. I., Donich, O. A., & Kutsyi, A. V. (2023). Klimat Kyieva ta yoho okolyts’ [Climate of Kyiv and its surroundings]. Varto, Kyiv (in Ukrainian).

Wyrzykiewicz-Raszewska, M. (2011). Phenology of Epipactis palustris (L.) Crantz growing in the wild and in an experimental garden. Roczniki Akademii Rolniczej w Poznaniu. Botanika-Steciana, 15, 73–80.

Xu, X., Du, X., Wang, F., Sha, J., Chen, Q., Tian, G., Zhu, Z., Ge, S., & Jiang, Y. (2020). Effects of potassium levels on plant growth, accumulation and distribution of carbon, and nitrate metabolism in apple dwarf rootstock seedlings. Frontiers in Plant Science, 11, 904.

Zaimenko, N. V. (Ed.). (2021). Suchasni metody v alelopatychnykh doslidzhenniakh [Modern methods in allelopathic research]. Lira-K, Kyiv (in Ukrainian).

Zaimenko, Н., Didyk, Н., Ellanska, Н., Rositska, Н., Kharytonova, І., & Yunosheva О. (2021). Implementation of modern technologies to alleviate soil sickness in urban green areas. Science and Innovation, 17(1), 64–77.

Zaіmenko, N., Gnatiuk, A., Gritsenko, V., Zakrasov, O., Pavliuchenko, N., Kharytonova, I., Dziuba, O., Didyk, N., Yunosheva, O., Blum, O., Likhanov, A., & Holichenko, N. (2024). Biochemical and allelopathic features of Adonis vernalis, Allium ursinum, and Leucojum vernum in the M. M. Gryshko National Botanical Garden of the NAS of Ukraine. Plant Introduction, 101–102, 3–18.