Društvo biologov Slovenije 2025 Vol. 68 | Št. 1 2 Acta Biologica Slovenica, 2025, 68 (1) Acta Biologica Slovenica, 2025, 68 (1) Založila/Published by Založba Univerze v Ljubljani / University in Ljubljana Press Društvo biologov Slovenije / Slovenian biological society Za založbo/For the publisher Gregor Majdič, rektor Univerze v Ljubljani / the Rector of the University of Ljubljana Anita Jemec Kokalj, predsednica Društva biologov Slovenije / Chairman of Slovenian Biological Society Izdala/Issued by Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za biologijo / University of Ljubljana, Biotehnical Faculty, Department of Biology Za izdajatelja/For the Issuer Marina Pintar, dekanja Biotehniške fakultete UL / Dean of Biotechnical Faculty Naslov uredništva/Editorial Office Address Univerza v Ljubljani, Biotehniška fakulteta, Acta Biologica Slovenica, Večna pot 111, 1000 Ljubljana, Slovenija Glavni urednik/Editor-in-chief Matevž Likar, Slovenija / Slovenia, matevz.likar@bf.uni-lj.si Odgovorna urednica/Managing editor Anita Jemec Kokalj, Slovenija / Slovenia, anita.jemec@bf.uni-lj.si Uredniški odbor/Editorial Board Gregor Belušič (SLO), Univerza v Ljubljani, Biotehniška fakulteta Tina Eleršek (SLO), Nacionalni inštitut za biologijo Božo Frajman (A), Univerza v Innsbrucku Alenka Gaberščik (SLO), Univerza v Ljubljani, Biotehniška fakulteta Király Gergely (HU), University of Sopron, Faculty of Forestry Gordana Glavan (SLO), Univerza v Ljubljani, Biotehniška fakulteta Katarina Hančević (HR), Institute for Adriatic Crops and Karst Reclamation Margit Heinlaan (EST), National Institute of Chemical Physics and Biophysics Georg A. Janauer (A), University of Vienna Vida Jojić (SRB), Univerzitet u Beogradu, Institut za biološka istraživanja „Siniša Stanković” Tina Klenovšek (SLO), Univerza v Mariboru, Fakulteta za naravoslovje in matematiko Dana Kühnel (GER), Helmholtz Centre for Environmental Research GmbH - UFZ Alenka Malej (SLO), Nacionalni inštitut za biologijo Nataša Mori (SLO), Nacionalni inštitut za biologijo Polona Mrak (SLO), Univerza v Ljubljani, Biotehniška fakulteta Maria Mueller (A), University of Salzburg Siniša Ozimec (HR), Univerza Josipa Juraja Strossmayerja Hubert Potočnik (SLO), Univerza v Ljubljani, Biotehniška fakulteta Tomislav Radić (HR), Institute for Adriatic Crops and Karst Reclamation Simona Strgulc Krajšek (SLO), Univerza v Ljubljani, Biotehniška fakulteta Mihael Jožef Toman (SLO), Univerza v Ljubljani, Biotehniška fakulteta Miloš Vittori (SLO), Univerza v Ljubljani, Biotehniška fakulteta Oblikovanje/Design Ajda Fortuna Naslovnica/Cover page Armadillo officinalis, avtor/author: Miloš Vittori To delo je ponujeno pod licenco Creative Commons Priznanje avtorstva-Deljenje pod enakimi pogoji 4.0 Mednarodna licenca (izjema so fotografije). / This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License (except photographies). Izdajanje revije sofinancira Javna agencija za znanstvenoraziskovalno in inovacijsko dejavnost Republike Slovenije (ARIS) The journal is co-financed by Slovenian Research and Innovation Agency (ARIS) Publication is free of charge. ISSN 1854-3073 (spletna verzija/online version) UDK 57(497.4) DOI: 10.14720/abs.68.1 http://journals.uni-lj.si/abs/ Acta Biologica Slovenica je indeksirana v – is indexed in: CAB Abstracts, Web of Science Clarivate 3 Acta Biologica Slovenica, 2025, 68 (1) Original Research Paper 4 Macrophyte Diversity and Ecosystem Services in Wetlands of Eastern Ranchi: A Multi-site Analysis of Abundance, Frequency, and Importance Value Index Patterns / Raznolikost makrofitov in ekološke funkcije v mokriščih vzhodnega Ranchija: Analiza vzorcev številčnosti, pogostosti in indeksa vrednosti pomembnosti na več lokacijah Jitendra Mahato, Sujit Ghosh 23 The effectiveness of teaching human evolution to 14- to 15-year-olds in Slovenia / Učinkovitost poučevanja evolucije človeka pri 14-do 15-letnikih v Sloveniji Jelka Strgar 33 Unveiling the antibacterial potential of latex-derived phytocomponents from Calotropis gigantea targeting bacterial Penicillin-binding proteins: chemical profiling, in silico docking, and in vitro lab validation / Razkritje antibakterijskega potenciala iz lateksa pridobljenih fitokomponent iz Calotropis gigantea, usmerjenih proti bakterijskim proteinom, ki vežejo penicilin: kemijsko profiliranje, in silico docking in in vitro potrditev v laboratoriju Arun Dev Sharma, Amrita Chauhan 45 Qualitative and Quantitative Detection of Virulence- Associated Genes in Escherichia Coli Isolates from Women with Urinary Tract Infections / Uporaba klasičnega PCR in RT-PCR za odkrivanje nekaterih genov faktorjev virulence bakterij Escherichia coli, izoliranih iz pacientk z uroinfektom Zainab Hussein Mahdi, Lina Abdulameer S. Alsaadi, Zainab Amer Hatem, Saade Abdalkareem Jasim 52 Impact of water deficit on the anatomical structure of more productive and less productive cashew trees (Anacardium occidentale L.) in Côte d’Ivoire / Vpliv pomanjkanja vode na anatomsko strukturo bolj produktivnih in manj produktivnih dreves indijskega oreščka (Anacardium occidentale L.) v Slonokoščeni obali Ky Abdoul Rahim Falk, Konaté Mory Latif, Kouamé N’Guessan François 69 Significant records of plants, algae, fungi, and animals in SE Europe and adjacent regions, 3 Aljaž Jakob, Laura Štampar, Žan Lobnik Cimerman, Simona Strgulc Krajšek, Miloš Vittori, Dren Dolničar 78 Cellulase, xylanase, lipase, and protease activities of selected wild mushrooms from Slovenia / Celulazne, ksilanazne, lipazne in proteazne aktivnosti nekaterih samoniklih slovenskih gob Larisa Lara Popošek, Luka Šparl, Ivona Pleše, Matija Hrovatin, Drejc Flajnik, Kristina Sepčić, Matej Skočaj 89 Phytochemical composition and antimicrobial activity of essential oil and extracts from the leaves of Peganum harmala L. in southern Algeria. / Fitokemična sestava in protimikrobna aktivnost eteričnega olja in izvlečkov iz listov Peganum harmala L. v južni Alžiriji. Ghillace Abderrahmane, Chouitah Ourida, Kiari Fatima Zohra, Fergoug Zineb, Daikh Zineeddine 104 Soil mesofauna diversity in agricultural systems of Slovenia using the QBS index and its modifications / Raziskava raznovrstnosti talne mezofavne v kmetijskih ekosistemih Slovenije z uporabo QBS indeksa in njegovih izpeljank Vid Naglič, Nataša Šibanc, Tine Grebenc, Irena Bertoncelj Review 118 Designer cellulosomes – catalytic nanomachines with significant potential in biotechnology and circular economy / Sintetični celulosomi – katalitični nanostroji z velikim potencialom v biotehnologiji in krožnem gospodarstvu Maša Vodovnik News 126 5. slovensko posvetovanje mikroskopistov Table of Contents 4 1 Research Scholar, Department of Botany, Sidho-Kanho-Birsha University, Purulia-723104, West Bengal, India 2 Associate Professor, Department of Botany, J.K. College, Purulia-723101, West Bengal, India * Corresponding author: E-mail address: sujit@jkcprl.ac.in Citation: Mahato, J., Ghosh, S., (2024). Macrophyte Diversity and Ecosystem Services in Wetlands of Eastern Ranchi: A Multi-site Analysis of Abundance, Frequency, and Importance Value Index Patterns. Acta Biologica Slovenica 68 (1) Received: 01.09.2024 / Accepted: 21.10.2024 / Published: 28.10.2024 https://doi.org/10.14720/abs.68.01.19686 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Macrophyte Diversity and Ecosystem Services in Wetlands of Eastern Ranchi: A Multi-site Analysis of Abundance, Frequency, and Importance Value Index Patterns Jitendra Mahato 1, Sujit Ghosh 2* Abstract Macrophytes, visible aquatic and semi-aquatic plants, are integral components of wetland ecosystems, playing crucial roles in maintaining biodiversity, providing ecosystem services, and ensuring ecological stability. This research examines the distribution, abundance, frequency, diversity, and ecosystem services of macrophytes in several wetlands (W1-W7) located in the eastern part of Ranchi district, Jharkhand, India. The study sites include Bundu Lake, Hindalco Pond, Choga Bada Talab, Raja Bandh, Kita Uparbandh, Tamar Bada Talab, and Rukka Dam. A quantitative assessment using quadrat sampling and phytosociological methods was conducted from September 2022 to March 2023 to ensure accurate data collection and analysis. The study documented 78 macrophyte species belonging to 33 families and 58 genera. Emergent macrophytes were found to be the dominant life form, constituting 69% of the recorded species. Several species demonstrated high frequency and abundance across multiple study sites, including Pontederia crassipes Mart., Alternanthera philoxeroides (Mart) Griseb., Hydrilla verticillata (L.f.) Royle, Ipomoega aquatica Forssk., and Nymphoides hydrophyllum (Lour.) kuntze. To assess the diversity and ecological characteristics of the macrophyte communities, various diversity indices were calculated. These included the Shannon-Wiener index, Simpson's index, Margalef index, and Pielou's Evenness index. The results of these analyses revealed high levels of macrophyte diversity and evenness within the studied wetlands. Twenty- one macrophytes were identified as key species and nine species as rare species based on their IVI (Importance Value Index) scores. The key species were found to provide essential ecosystem services, including erosion control, water quality improvement, nutrient cycling, carbon sequestration, and habitat provision for other organisms. This research contributes significantly to our understanding of macrophyte diversity patterns and traits-based ecosystem services in wetland 5 Acta Biologica Slovenica, 2025, 68 (1) Raznolikost makrofitov in ekološke funkcije v mokriščih vzhodnega Ranchija: Analiza vzorcev številčnosti, pogostosti in indeksa vrednosti pomembnosti na več lokacijah Izvleček Makrofitiso sestavni del mokriščnih ekosistemov, saj imajo ključno vlogo pri ohranjanju biotske raznovrstnosti, zagotavljanju ekosistemskih storitev in zagotavljanju ekološke stabilnosti. Ta raziskava preučuje razširjenost, številčnost, pogostost, raznolikost in ekološko funkcijo makrofitov v več mokriščih (W1-W7), ki se nahajajo v vzhodnem delu okrožja Ranchi v Džarkhandu v Indiji. Študijska območja vključujejo jezero Bundu, ribnik Hindalco, Choga Bada Talab, Raja Bandh, Kita Uparbandh, Tamar Bada Talab in jez Rukka. Kvantitativna ocena z uporabo kvadratnega vzorčenja in fitosocioloških metod je potekala od septembra 2022 do marca 2023, s čimer smo želeli zagotovitinatančnost zbranih podatkov in analiz. V študiji je bilo dokumentiranih 78 vrst makrofitov, ki pripadajo 33 družinam in 58 rodovom. Ugotovljeno je bilo, da so prevladujoča življenjska oblika emergentni makrofiti, ki so predstavljali 69 % zabeleženih vrst. Več vrst je pokazalo visoko pogostost in številčnost na več študijskih območjih, vključno s Pontederia crassipes Mart, Alternanthera philoxeroides (Mart) Griseb, Hydrilla verticillata (L.f.) Royle, Ipomoea aquatica Forssk in Nymphoides hydrophyllum (Lour.) kuntze. Za oceno raznolikosti in ekoloških značilnosti združb makrofitov so bili izračunani različni indeksi raznolikosti. Med njimi so bili Shannon-Wienerjev indeks, Simpsonov indeks, Margalefov indeks in Pieloujev indeks enakomernosti. Rezultati teh analiz so pokazali visoko stopnjo raznolikosti in enakomernosti makrofitov v preučevanih mokriščih. Enaindvajset makrofitov je bilo opredeljenih kot ključne vrste, devet vrst pa kot redke vrste na podlagi njihovih ocen IVI (Importance Value Index). Ugotovljeno je bilo, da ključne vrste zagotavljajo bistvene ekosistemske storitve, vključno z nadzorom erozije, izboljšanjem kakovosti vode, kroženjem hranil, sekvestracijo ogljika in zagotavljanjem habitatov za druge organizme. Ta raziskava pomembno prispeva k razumevanju vzorcev raznolikosti makrofitov in ekosistemskih storitev, ki temeljijo na lastnostih, v mokrotnih ekosistemih. Ugotovitve te študije imajo pomembne posledice za razvoj in izvajanje učinkovitih strategij ohranjanja teh ključnih mokriščnih ekosistemov v vzhodnem Ranchiju (W1- W7). Ključne besede ekološka vloga, ključne vrste, makrofiti, kvantitativna ocena, mokrišče ecosystems. The findings of this study have important implications for the development and implementation of effective conservation strategies for these vital wetland ecosystems in the eastern Ranchi (W1- W7). Keywords Ecosystem services, key species, macrophytes, quantitative assessment, wetland Introduction Wetlands are the most vital ecosystems on our planet, offering a wide range of ecological services such as water purification, flood control, carbon storage, and the conservation of biodiversity. These ecosystems help regulate water cycles and are rich sources of biodiversity, supporting numerous species, genetic variations, and diverse ecosystems (Mitsch et al., 2015; Shiji et al., 2016). Among the key players in these environments are macro- phytes, large aquatic plants that you can see with the naked eye. Macrophytes contribute significantly to the health of aquatic ecosystems by promoting sediment deposition, carbon capture, water filtration, and the removal of pol- lutants. Additionally, they serve as sources of bioenergy biochar and help mitigate the impacts of climate change. 6 Acta Biologica Slovenica, 2025, 68 (1) Beyond these environmental roles, macrophytes provide natural medicines, fibres, and other resources that benefit human society, reinforcing the link between biodiversity and human well-being (Kumar et al., 2023). Unfortunately, these vital plants are increasingly under threat. Pollution, habitat destruction, invasive species, and climate change are all taking a toll on macrophyte populations, leading to degraded ecosystems and a loss of biodiversity (Lesiv et al., 2020). Human activities, such as unsustainable water man- agement, overharvesting, sediment build-up, and the use of chemicals, further compound these challenges, damaging both macrophytes and the essential services they provide. In the eastern part of Ranchi district, Jharkhand, India, several wetlands serve as important habitats for a variety of macrophyte species. Wetlands like Bundu Lake, Hindalco Pond, Choga Bada Talab, Raja Bandh, Kita Uparbandh, Tamar Bada Talab, and Rukka Dam host a diverse range of aquatic vegetation. These plants play a crucial role in main- taining the health and functioning of these ecosystems. Understanding how these macrophytes are distributed, how abundant they are, how frequently they appear, and what services they offer to the ecosystem is essential if we are to develop effective conservation and management strategies. Various studies have looked at macrophyte diversity and the ecological significance of wetlands in other regions (Swamy et al., 2016; Xu et al., 2019; Bhanja et al., 2023). In the Chota Nagpur Plateau, where Ranchi district is located, researchers have explored macrophyte diversity in places like Ranchi Lake and Kanke Dam. For instance, Lal and Lal (2021) found 36 macrophyte species in Kanke Dam and 17 species in Ranchi Lake. Other stud- ies have mentioned the macrophyte diversity in various wetlands across the Ranchi district in their broader floristic surveys (Verma and Pandeya, 2008; Mukherjee and Kumar, 2020). However, most of these studies focus on individual sites and don't provide a broader picture of species distri- bution, abundance, or diversity across multiple wetlands. Although macrophytes are well known for their role in delivering ecosystem services (Thomaz, 2023), there is a lack of site-specific information in the wetlands of the east- ern Ranchi region that connects species composition to ecosystem functions. Furthermore, the lack of precise sug- gestions based on in-depth ecological data in the region's current conservation strategies for wetlands impairs the development of efficient management strategies. Comprehensive research on the distribution, abun- dance, frequency, diversity, and ecosystem services of macrophytes in the seven selected wetlands in the eastern portion of Ranchi district is missing despite the ecological relevance of wetlands and the value of these plants. The lack of comparative study of species frequency and diversity indices, the scarcity of data on specific species distributions and abundances, and the lack of knowledge on ecosystem services are some of the major research gaps. To fill these gaps, a thorough investigation of the biological patterns and ecosystem services provided by the macrophyte species in these wetlands is necessary. The primary goal of this research is to explore the diversity of macrophytes, their abundance and distribution patterns, and the ecosystem services they provide across seven selected wetlands in the eastern Ranchi district. Materials and Methods The study area The study was carried out in Ranchi District, the capital of Jharkhand, India, which is located between 85°15′-85°23′ E longitude and 23°21′-23°87′ N latitude, spanning 7,698 square kilometres of steep terrain. The district is located in the southern section of the Chota Nagpur Plateau, an extension of the Deccan Plateau, and is bordered to the north by Ramgarh, Hazaribagh, and Chatra, to the south by Khunti, to the west by Latehar, Lohardaga, and Gumla, and to the east by Saraikela and Purulia (West Bengal). Ranchi has a humid subtropical climate, with summer tem- peratures ranging from 20-40°C and winter temperatures ranging from 1-25°C, and it receives both seasonal and cyclonic rainfall (Rani et al., 2011). The Subarnarekha and its tributaries form the principal river system. Selection of Wetlands For the quantitative analysis of wetland biodiversity and ecological conditions, seven wetlands from the eastern region of the district were selected, each representing dif- ferent blocks. The wetlands include Bundu Lake (W1), Hin- dalco Pond (W2), Choga Bada Talab (W3), Raja Bandh (W4), Kita Upar Bandh (W5), Tamar Bada Talab (W6), and Rukka Dam (W7) (Fig. 1). Table 1 provides general information about these selected wetlands, including their geograph- ical coordinates, elevation, surface area, and macrophyte cover. The selection of these wetlands aimed to capture 7 Acta Biologica Slovenica, 2025, 68 (1) both urban and rural settings, allowing for a comparative analysis of human influence and ecological health across varying landscapes. The catchment areas and corresponding human influ- ences on these wetlands were assessed to understand their ecological status. W1 (Bundu Lake), an urban wetland covering 40.47 hectares, is located near markets, human settlements, and agricultural land. It experiences heavy pol- lution due to urban sewage, domestic waste, agricultural runoff, and shrinkage caused by human settlement expan- sion, road adjacency, and recreational use. Similarly, W2 (Hindalco Pond), a smaller 2.43-hectare urban wetland near Muri Station, is significantly impacted by industrial activities and urban settlements. This wetland faces severe pollution from industrial and domestic sewage, siltation, and indus- trial encroachment. In contrast, W3 (Choga Bada Talab), a rural wetland spanning 10.93 hectares, is surrounded by agricultural land and open grasslands. Its pollution mainly stems from agricultural runoff, domestic use, and irrigation, but it is less affected by human interference compared to the urban wetlands. Another rural wetland, W4 (Raja Bandh), covering 9.41 hectares, has a catchment area consisting of hilly uplands, agricultural fields, and open grasslands. Pollution sources in this wetland include road construc- tion, domestic activities, cattle bathing, and the partial conversion of land for agriculture. W5 (Kita Uparbandh), an urban wetland of 2.02 hectares, is bordered by natural hilly vegetation, agricultural land, and a major road. It suffers from pollution caused by domestic sewage, proximity to human settlements, road adjacency, and shrinkage due to increasing urbanization. W6 (Tamar Bada Talab), a rural wet- land of 5.67 hectares, is less affected by human activities, with catchment areas consisting of open grassland and agricultural land. Pollution in this wetland is primarily due to agricultural runoff and limited domestic use, making it one of the least impacted wetlands in the study. Lastly, W7 (Rukka Dam) is a riverine, natural wetland with minimal human influence, spanning a large rural area (10199.77 hectares). Its catchment areas include agricultural land, open grasslands, and forested regions, with no major anthropogenic disturbances. Pollution in W7 is limited to agricultural runoff and occasional recreational activities, maintaining its status as a relatively pristine ecosystem. The variety of settings across these seven wetlands allows for a comprehensive analysis of how human activities influence wetland biodiversity and ecological conditions. Figure 1. Location map of the study area with designated sampling sites (W1-W7) (Source: Mishra and Lal, 2023 and modified based on the location of wetlands). Slika 1. Lokacijska karta območja študije z označenimi mesti vzorčenja (W1-W7) (Vir: Mishra in Lal, 2023 in spremenjeno na podlagi lokacije mokrišč). 8 Acta Biologica Slovenica, 2025, 68 (1) Sampling methodology Fieldwork was conducted from September 2022 to March 2023, spanning the post-monsoon to winter seasons. Quadrat sampling was used to assess macrophytes qual- itatively and quantitatively. Ten 1 m × 1 m quadrats were randomly placed throughout each wetland, with three replicates per site. Sampling was carried out up to 2 m from the wetland's periphery to its centre. The macrophytes that occurred or fell within each sampling unit were counted by hand and listed by species (Margalef, 1969). Identification and nomenclature Plant species were identified using standard protocols, work and keys provided by the books of 'Aquatic and Wetland Plants of India' (Cook 1996) and 'Wetland Flora of West Bengal' (Chowdhury and Chowdhury 2022). Valid scientific names were cross-checked using the Plants of the World Online (POWO 2024) and World Flora Online (WFO 2024) databases. Voucher samples of identified macrophytes were prepared and deposited at the Botany Department herbarium of Sidho-Kanho-Birsha University (SKBU), Purulia, West Bengal, India, for future reference and verification. Data analysis The phytosociological characteristics were determined using established procedures (Margalef, 1969). The diver- sity indicators, including Shannon-Wiener, Simpson's, spe- cies richness, and species evenness, were calculated using Magurran's (2004) methods. The number of individuals in each species was counted to calculate phytosociological measures such as percent frequency, frequency class (Raunkiaer, 1934), relative frequency, abundance, density, relative density, and IVI (Importance Value Index) using the following formulas. Statistical analyses Microsoft Excel was used to conduct statistical analyses comparing macrophyte diversity and other factors among the seven wetlands (W1 to W7). The data was interpreted by the application of descriptive statistics and comparative analyses. Name of the Wetlands, (Blocks). Latitude Longitude Altitude Size hectares Existing Sp. Bundu Lake. (Bundu). 23.1614 N 85.586489 E 259.47 met. 40.47 ha. 31 Hindalco Pond. Muri, (Silli). 23.38126 N 85.867212 E 198.63 met. 2.43 ha. 25 Choga Bada Talab. Choga, (Sonahatu). 23.21299 N 85.7433049 E 190.09 met. 10.93 ha. 27 Raja Bandh. Rahe, (Rahe). 23.24986 N 85.6375088 E 251.66 met. 9.41 ha. 27 Kita Uparbandh. Kita, (Angara). 23.36492 N 85.765632 E 251.55 met. 2.02 ha. 25 Tamar Bada Talab. Tamar, (Tamar). 23.05430 N 85.65870 E 189.46 met. 5.67 ha. 29 Rukka Dam, (Ormanjhi, only Rukka portion.) 23.43663 N 85.462674 E 596 met. 10199.77 ha. 39 Table 1. Overview of the selected wetlands. Tabela 1. Pregled izbranih mokrišč. Frequency (%) = Total number of quadrants in which species has occurred x 100 Total number of quadrants studied Relative Frequency = Frequency of a particular species x 100 Sum of frequencies of all species Abundance = Total number of indivisuals of the species Total number of quadrants in which the species has occurred Relative Abundance = Abundance of a particular species x 100 Total abundance of all species Density = Total number of indivisuals of the species Total number of quadrants studied Relative Density = Density of a particular species x 100 Total density of all species IVI = (Relative Frequency + Relative Abundance + Relative Density). 9 Acta Biologica Slovenica, 2025, 68 (1) Results Macrophyte Species Distribution and Abundance Across the seven wetlands (W1 to W7), a total of 78 mac- rophyte species (Table 2) from 33 families and 58 genera were recorded. Emergent macrophytes dominated the flora, making up 69% of the total, followed by rooted floating-leaf species (12%), submerged species (11%), and free-floating species (8%). Emergent macrophytes were the most prevalent, with 54 species recorded, including Cyperus iria L. particularly abundant in Wetland W5, Paspalum distichum L. in Wet- lands W3 and W5, and Echinochloa colonum (L.) Link. in Wetlands W4, W5, and W7. Rooted with floating-leaved macrophytes were represented by nine species, with Nymphaea pubescens Willd., Nymphoides hydrophyllum (Lour.) kuntze, and Trapa natans var. bispinosa (Roxb.) Makino. being the most common. Free-floating macro- phytes comprised six species, with Lemna perpusilla Torr. and Spirodela polyrhiza (L.) Schleid. frequently observed in Wetlands W1, W2, W6, and W7 (Table 2). Lastly, nine species of submerged macrophytes were documented, with Potamogeton crispus L. and Potamogeton nodosus Poir. dominating across the study sites. Scientific Name IVI W1 W2 W3 W4 W5 W6 W7 G.F. Acmella paniculata (Wall ex DC.) R.K. Jansen 3.12 9.58 7.16 4.72 E Actinoscirpus grossus (L.f.) Goetgh. & D.A. Simpson 7.01 E Aeschynomene indica L. 7.36 4.17 E Alternanthera philoxeroides (Mart) Griseb. 18.75 14.41 19.69 13.97 12.30 E Alternanthera sessilis (L.) DC. 11.71 11.61 13.65 14.36 9.80 7.86 E Ammannia baccifera L. 6.17 E Azolla pinnata R.Br. 11.74 20.54 11.63 7.28 FF Bacopa monnieri (L.) Wettst. 17.76 E Centella asiatica (L.) Urb. 4.75 6.47 E Ceratophyllum demersum L. 7.59 S Ceratopteris thalictroides (L.) Brongn. 4.29 3.84 E Chenopodium ficifolium Sm. 8.31 E Colocasia esculenta (L.) Schott 3.86 7.16 5.01 4.54 2.87 E Cyperus brevifolius (Rottb.) Hassk. 7.56 E Cyperus compressus L. 10.40 14.52 12.55 13.25 10.75 E Cyperus difformis L. 10.04 12.62 17.81 10.03 E Cyperus flavidus Retz. 7.99 E Cyperus fuscus L. 9.00 E, Cyperus iria L. 14.25 17.94 E Cyperus polystachyos Rottb. 5.98 E Dentella repens (L.) J.R. Forst. & G. Forst. 13.42 E Echinochloa colonum (L.) Link 15.10 16.24 11.18 E Eclipta prostrata (L.) L. 7.03 9.39 7.66 9.29 9.44 7.68 6.05 E Table 2. Distribution of macrophyte species with IVI and growth forms. Tabela 2. Razporeditev vrst makrofitov z IVI in oblikami rasti. 10 Acta Biologica Slovenica, 2025, 68 (1) Eleusine indica (L.) Geartn. 12.51 9.49 E Evolvulus nummularius (L.) L. 7.75 12.62 5.13 E Fimbristylis aestivalis (Retz.) Vahl 9.51 E Fimbristylis alboviridis C.B. Clarke 12.95 E Fimbristylis argentea (Rottb.) Vahl 15.62 E Fimbristylis bisumbellata (Forssk.) Bubani 11.10 17.28 12.34 E Fuirena ciliaris (L.) Roxb. 10.55 17.09 10.03 8.76 E Grangea maderaspatana (L.) Poir. 8.67 E Heliotropium indicum L. 4.39 9.26 E Hydrilla verticillata (L.f.) Royle 18.15 15.88 18.29 S Hygrophila auriculata (Schumach.) Heine 9.01 E Ipomoea aquatica Forssk. 9.84 12.98 10.84 10.16 11.90 8.76 7.54 RFL Ipomoea carnea Jacq. 6.69 6.68 7.01 9.26 5.46 3.10 E Kyllinga melanosperma Nees 12.05 E Lemna perpusilla Torr. 17.57 24.81 15.48 10.12 FF Lindernia parviflora (Roxb.) Haines 11.77 E Limnophila sessiliflora (Vahl) Blume 14.25 S Ludwigia adscendens (L.) H. Hara 9.09 8.05 8.03 RFL Ludwigia perennis L. 4.57 9.31 5.07 5.01 3.63 E Louisiella paludosa (Roxb.) Landge 13.65 E Marsilea minuta L. 18.99 15.42 11.03 16.34 14.62 8.16 RFL Melilotus indicus (L.) All. 7.16 E Nelumbo nucifera Geartn. 3.58 6.81 8.65 RFL Nymphaea nouchali Burm.f. 3.69 3.67 RFL Nymphaea pubescens Wild. 5.45 4.48 7.08 8.70 12.74 10.81 RFL Nymphoides hydrophyllum (Lour.) Kuntze 22.84 20.23 12.39 RFL Nymphoides indica (L.) Kuntze 12.80 p 13.43 RFL Oldenlandia corymbosa L. 8.46 9.51 8.58 5.15 E Ottelia alismoides (L.) Pers. 6.68 S Panicum repens L. 12.95 21.07 17.32 13.53 10.83 E Paspalum distichum L. 16.73 16.99 16.81 12.76 E Persicaria barbata (L.) H. Hara 3.44 E Persicaria hydropiper (L.) Delabre 7.16 10.81 E Persicaria glabra (Willd.) M.Gomez 5.62 3.16 E Phyla nodiflora (L.) Greene 9.16 5.79 E Pistia stratiotes L. 10.22 15.15 FF Polygonum plebeium R.Br. 14.52 12.62 7.28 E Pontederia crassipes Mart. 20.72 21.91 17.20 FF 11 Acta Biologica Slovenica, 2025, 68 (1) Pontederia vaginalis Burm.f. 6.68 4.18 E Potamogeton crispus L. 16.15 9.13 S Potamogeton nodosus poir. 11.28 9.24 S Pouzolzia zeylanica (L.) Benn. 4.48 E Ranunculus sceleratus L. 6.68 E Rotala rotundifolia (Buch. -Ham. ex Roxb.) Koehne 17.07 9.97 E Salvinia cucullata Bory 9.48 FF Schoenoplectiella articulata (L.) Lye. 5.62 E Schoenoplectiella senegalensis (Steud.) Lye 6.17 4.86 E Scleromitrion brachypodum (DC.) T.C. Hsu 8.35 E Spirodela polyrhiza (L.) Schleid. 17.20 23.26 16.44 8.84 FF Torenia crustacea (L.) Chan & Schltdl. 9.08 7.35 7.88 6.71 E Trapa natans var. bispinosa (Roxb.) Makino 6.51 15.98 14.56 RFL Typha domingensis Pers. 9.58 4.58 E Utricularia aurea Lour. 8.98 4.85 S Utricularia stellaris L. f. 6.64 7.72 7.90 9.19 5.87 5.97 S Vallisneria spiralis L. 9.75 7.34 6.00 S (Abbreviations: G. F= Growth form, E = Emergent, S = Submerged, RFL = Rooted with floating leaves, FF = Free floating.) Dominant families To determine the dominant families across the study sites, a quantitative approach was employed. The proportion of species represented by each family was calculated by dividing the number of species within a particular family by the total number of species across all families. This proportion was then expressed as a percentage using the following formula: The family with the highest percentage was identified as the dominant family, representing the largest contribution to the overall macrophyte diversity in the wetlands. This approach allowed for a clear assessment of the relative importance of each family in the macrophyte community. The Cyperaceae family was the most dominant based on species richness and percentage representation, with 16 species accounting for 20.51% of the total species. Other notable families included Poaceae (6.41%), Araceae (5.12%), Polygonaceae (5.12%), Hydrocharitaceae (3.85%), Convolvulaceae (3.85%), Asteraceae (3.85%), and Rubiaceae (3.85%). These families made significant contri- butions to the overall macrophyte diversity in the wetlands (Fig. 2). Frequency and Distribution of Macrophyte Species The frequency of occurrence of various plant species across different wetlands varied considerably. Species such as Pontederia crassipes Mart. and Alternanthera philoxeroi- des, Hydrilla verticillata exhibited high frequency across various wetlands, suggesting adaptability to a range of envi- ronmental conditions. Nymphoides hydrophyllum showed a particularly high frequency in Wetland 3 (W3) and Wetland 4 (W4), with occurrences of 90% and 83.33%, respectively. Conversely, low-frequency species like Acmella paniculata, Pouzolzia zeylanica (L.) Benn., Persicaria glabra (Willd.) M. Gómezand Torenia crustacea (L.) Chan & Schltdl. were restricted to specific areas, likely due to their sensitivity to habitat modifications (see Table 3 for details). Percentage of Family = Number of Species in a Particular Family x 100 Total number of Species across all Families 12 Acta Biologica Slovenica, 2025, 68 (1) Figure 2. Distribution of dominant families across the study sites (W1-W7). Slika 2. Porazdelitev dominantnih družin na raziskovalnih območjih (W1-W7). High Frequency Species Frequency (%) Wetlands High Frequency Species Frequency (%) Wetlands Pontederia crassipes 85.71%, 70%, 81.25% W1, W2, W7 Acmella paniculata 14.29% W1 Alternanthera philoxeroides 64.29%, 62.5%, 62.5% W1, W5, W7 Torenia crustacea 14.29%, 20% W1, W2 Alternanthera sessilis 50%, 50% W1, W5 Chenopodium ficifolium 10% W2 Hydrilla verticillata 57.14%, 70% W1, W6 Pouzolzia zeylanica 10% W2 Ipomoea aquatica 50%, 50%, 43.75% W2, W3, W7 Bacopa monnieri 20% W3 Nymphaea pubescens 60%, 62.5% W6, W5 Persicaria glabra 20%, 18.75% W3, W7 Panicum repens 70% W2 Persicaria hydropiper 16.67%, 20% W4, W2 Pistia stratiotes 81.5% W7 Ammannia baccifera 16.67% W4 Trapa natans var. bispinosa 60%, 58.33% W6, W4 Fimbristylis alboviridis 16.67% W4 Ludwigia adscendens 62.5%, 50%, 50% W7, W3, W6 Grangea maderaspatana 16.67% W4 Nymphoides hydrophyllum 90%, 83.33% W3, W4 Aeschynomene indica 12.5% W5 Nymphoides indica 50%, 50% W2, W6 Centella asiatica 12.5% W5 Potamogeton crispus 66.67% W4 Ceratopteris thalictroides 12.5%, 25% W7, W5 Salvinia cucullata 50% W7 Colocasia esculenta 18.75%, 20% W7, W6 Cyperus difformis 50% W5 Azolla pinnata 10% W6 Lemna perpusilla 10% W6 Spirodela polyrhiza 10% W6 Table 3. Frequency and distribution of macrophyte species. Tabela 3. Pogostost in porazdelitev vrst makrofitov. 13 Acta Biologica Slovenica, 2025, 68 (1) Raunkiaer's Frequency Analysis The frequency distribution of plant species within each wetland was calculated using Raunkaier's law of frequency classes (1934). The results are presented in Table 4. By ana- lyzing and comparing them to Raunkaier's law of frequency classes (A > B > C ≥ D < E, where the value of the frequency class comprises class A= 53%, B=14%, C=9%, D= 8% and E=16%) across all wetlands (W1 - W7), significant insights were gained into the spatial distribution and diversity of macrophytes. This analysis highlights the varying patterns of species diversity, their spatial distribution, and the eco- logical status of these wetlands. Wetland W1 had the highest percentage in Class B (64.52%), indicating a heterogeneous vegetation structure, while Wetland W2 displayed higher disturbance with lower frequencies in Classes C and E (4.00% and 0.00%, respec- tively). In Wetland W3, Class B dominated with a frequency of 55.55%, indicating moderate and stable diversity, while Class D was absent. Wetland W4 also showed a strong presence of Class B at 48.15%, with Classes A and C equally represented at 22.22%. Wetland W5 exhibited Class B at 48.00% and Class C at 36.00%, suggesting a less diverse community structure. In Wetland W6, Class B was again the most frequent at 44.83%, with limited diversity overall. Wetland W7 recorded Class B at 41.02% and Class A at 38.46%, reflecting a consistent distribution pattern across the wetlands. Hence, the study exposed distinct patterns of species diversity and ecological dynamics within these wetland ecosystems. Diversity Indices and Evenness Analysis The current study investigates macrophyte diversity and evenness across the wetlands under study (W1-W7) using several established biodiversity indices (Table 5), including the Shannon and Weiner diversity index (H'), Simpson's index of diversity (1-D), Margalef diversity index, and Pielou's evenness index (J'). These indices provide insights into the composition, richness, and evenness of macro- phyte communities, offering a comprehensive view of their biodiversity status (Fig. 3). Study sites Class A Class B Class C Class D Class E Raunkaier's Frequency W1 9.68 64.52 19.36 3.22 3.22 A < B > C > D = E W2 52.00 36.00 4.00 8.00 0.00 A > B > C < D > E W3 22.22 55.55 18.53 0.00 3.70 A < B > C > D < E W4 22.22 48.15 22.22 3.70 3.70 A < B > C > D = E W5 8.00 48.00 36.00 8.00 0.00 A < B > C > D > E W6 34.48 44.83 17.24 3.45 0.00 A < B > C > D > E W7 38.46 41.02 10.26 5.13 5.13 A < B > C > D = E Index W1 W2 W3 W4 W5 W6 W7 Shannon and Weiner Diversity Index (H): 3.1903 2.9512 3.1383 3.1781 3.0264 3.2651 3.4968 Simpson's Index of Diversity (1- D): 0.9514 0.9367 0.9505 0.9545 0.9464 0.9542 0.9654 Margalef Diversity Index: 4.14533 3.853248 3.925082 3.887354 3.628268 4.207085 5.08069 Pielou's Evenness (J') 0.92904649 0.916832 0.952205 0.964278 0.940208 0.96965 0.954481 Table 4. Raunkaier's frequency classes (%) of the study sites (W1-W7). Tabela 4. Raunkaierjevi frekvenčni razredi (%) raziskovalnih območij (W1-W7). Table 5. Values of different diversity Indices and evenness. Tabela 5. Vrednosti različnih indeksov raznolikosti in enakomernosti. 14 Acta Biologica Slovenica, 2025, 68 (1) The Shannon-Weiner diversity index (H') revealed that Wetland W7 had the highest biodiversity (H' = 3.4968), indicating a well-diversified and evenly distributed species community. In contrast, Wetland W2 exhibited the lowest biodiversity (H' = 2.9512), suggesting reduced species rich- ness and evenness. Simpson's Index of Diversity (1-D), which also considers species richness and evenness, showed high values for wetlands W1, W3, W4, W6, and W7, with scores ranging from 0.9505 to 0.9654. These high values indicate that these wetlands have diverse and stable ecosystems. However, W2 and W5 had slightly lower (1-D) values (0.9367 and 0.9464), consistent with their lower H' scores, reflecting reduced biodiversity or dominance by certain species. The Margalef diversity index (DMg), which focuses on species richness, highlighted that W1 (4.14533), W3 (3.925082), W4 (3.887354), and W6 (4.207085) had rela- tively high species richness. Wetland W7 had the highest richness overall, while W5 exhibited the lowest species richness. The lower DMg values in W2 (3.853248) and W5 (3.628268) suggest that these wetlands experience reduced species richness, likely due to environmental stressors such as pollution and human activities. Pielou's Evenness index (J'), which measures species distribution and ecological balance, showed high evenness in wetlands W3, W4, W5, W6, and W7, indicating stable environmental conditions. Wetland W6 had the highest evenness score (0.96965), whereas W1 (0.92904) and W2 (0.91683) dis- played the lowest evenness, possibly due to the influence of environmental or anthropogenic factors. Categorization of IVI Values The analysis of IVI values across these wetlands (W1-W7) revealed distinct patterns in the distribution of species based on their ecological significance. The important value index (IVI) serves as an indicator of a species' role within the community, considering its abundance, frequency, and dominance. Table 6 illustrates the categorization and ranges of IVI scores for each study site, highlighting species with low, intermediate, and high IVI values. The percentage of species with low, intermediate, and high IVI values varied among the wetlands, highlighting the diverse ecological roles and dominance of macrophyte species. Species with low IVI values, such as Acmella paniculata, Figure 3. Comparative diversity indices of macrophytes at the study sites (W1-W7). (Shanon and Weiner, Simpson, Margalef, Pielou’s index). Slika 3. Primerjalni indeksi raznolikosti makrofitov na preučevanih območjih (W1-W7). (Shanon in Weiner, Simpson, Margalef, Pieloujev indeks) 15 Acta Biologica Slovenica, 2025, 68 (1) Hydrilla verticillata, Colocasia esculenta, exhibit low abun- dance, frequency, or dominance across wetlands W1–W7, indicating their modest roles in wetland structure and func- tioning. Intermediate IVI species, which dominate most of the wetlands (W1–W7), make up 58–77% of the total mac- rophyte community. These species, such as Alternanthera philoxeroides and Azolla pinnata, contribute significantly to the stability of wetland ecosystems by maintaining bio- diversity, habitat provisioning, and food supply for various organisms. High IVI species, like Pontederia crassipes and Nymphoides hydrophyllum are the most frequent and dominant, playing crucial roles in nutrient cycling, sediment retention, and habitat structuring. Rare Species Nine species were identified as rare based on their low relative density, frequency, dominance and importance value index (IVI). Significant rare species included Grangea maderaspatana (IVI 8.67) and Scleromitrion brachypodum (DC.) T.C. Hsu (IVI 8.35), both crucial for habitat provision and soil stabilization. These species were primarily found in W3 and W4 and are vulnerable to environmental changes and anthropogenic disturbances. Other rare species included Actinoscirpus grossus (IVI 7.01), Schoenoplectiella articulata (L.) Lye (IVI 5.62), Ranunculus sceleratus L. (IVI 6.68), Ammannia baccifera (IVI 6.16), Ottelia alismoides (L.) Pers. (IVI 6.68), Ceratopteris thalictroides (IVI 4.29) and Pouzolzia zeylanica (IVI 4.48). Trait-Based Ecosystem Services of Macrophytes In this study, 21 dominant macrophyte species were recog- nized as key species due to their high IVI, abundance, and density, contributing significantly to numerous ecosystem services (Table 7). These species include 52% emergent macrophytes, 10% submerged, 24% free-floating, and 14% rooted floating-leaved species. Traits such as extensive root systems, dense root hairs, and rhizome formation are vital for soil stabilization, nutrient absorption, and sediment retention in wetlands. Species like Alternanthera philoxeroides, Bacopa monnieri, Potamogeton crispus, Azolla pinnata, Lemna perpusilla are particularly efficient at trapping sediments, preventing erosion and preserving the structural integrity of wetlands. Their rapid root growth, deep rooting systems and rhizome formation further enhance their ability to stabilize sediments and promote overall wetland health through significant above-ground biomass production. Dense root networks, fibrous roots and fine root hairs play a key role in retaining sediments, contributing significantly to the resilience and stability of wetland ecosystems. In terms of water quality improvement, species such as Hydrilla verticillata, Lemna perpusilla, and Spirodela polyrhiza play key roles in nutrient uptake and pollutant absorption through root exudates. These species, along with Nymphoides hydrophyllum, Pontederia crassipes and Rotala rotundifolia contribute to sediment retention and Study sites Ranges of IVI Low IVI Score Intermediate IVI Score High IVI Score W1 3.12 - 20.72 Acmella paniculata (IVI-3.12) Azolla pinnata (IVI- 11.74) Pontederia crassipes (IVI- 20.72) W2 4.48 - 24.81 Nymphaea pubescens (IVI 4.48) Alternanthera philoxeroides (IVI 14.41) Lemna perpusilla (IVI – 24.81) W3 5.62 – 22.84 Persicaria glabra (IVI – 5.62) Cyperus iria (IVI – 14.25) Nymphoides hydrophyllum (IVI - 22.84) W4 3.67 – 20.23 Nymphaea nouchali (IVI – 3.67) Fimbristylis bisumbellata (IVI – 12.34) Nymphoides hydrophyllum (IVI – 20.23) W5 4.17 – 19.69 Aeschynomene indica (IVI – 4.17) Ipomoea aquatica (IVI – 11.90) Alternanthera philoxeroides (IVI – 19.69) W6 3.63 – 18.29 Ludwigia perennis (IVI – 3.63) Nymphaea pubescens (IVI- 10.81) Hydrilla verticillata (IVI – 18.29) W7 2.87 – 17.20 Colocasia esculenta (IVI - 2.87) Rotala rotundifolia (IVI- 9.97) Pontederia crassipes (IVI – 17.20) Table 6. Categorization and ranges of IVI scores of macrophytes in the study sites (W1-W7). Tabela 6. Kategorizacija in razponi ocen IVI makrofitov na preučevanih območjih (W1-W7). 16 Acta Biologica Slovenica, 2025, 68 (1) biomass production, creating surface area for gas exchange and improving water quality. For fodder production, species such as Alternanthera philoxeroides, Cyperus iria, Panicum repens, and Paspalum distichum, exhibit high biomass pro- duction and dense vegetation cover, making them valuable as palatable fodder for livestock while contributing to the stability and productivity of wetland ecosystems. Discussion The study found a diverse array of macrophyte species across these seven wetlands (W1-W7), with emergent plants dominating (69%), aligning with Vymazal's (2013) findings on free-water surface-constructed wetlands. The presence of all four growth forms indicates a complex ecosystem with diverse microhabitats crucial for supporting aquatic fauna and ecosystem functions (Jeppesen et al., 2012). The low percentage of submerged species (11%) might suggest water clarity or quality issues, as noted by Blindow et al. (2000). Localized dominant macrophytes play vital roles in wetland ecosystems. Species like Lemna perpusilla, Spirodela polyrhiza, Bacopa monnieri, and Azolla pinnata thrive in specific environmental conditions. L. perpusilla and S. polyrhiza prefer shallow, nutrient-rich waters with high light intensity and warm temperatures (Smith and Barko, 1990; Brock, 1997). B. monnieri adapts to various light conditions in shallow depths (Smith, 2014), while A. pinnata tolerates diverse water depths and can thrive in nutrient-poor waters due to its nitrogen-fixing ability Traits of the macrophytes Name of the macrophytes Soil erosion control, soil stabilization and nutrient absorption Extensive root system Root surface area Root hair density Root length, root depth Root exudates Mycorrhizal associations Alternanthera philoxeroides, Bacopa monnieri, Cyperus iria, Cyperus difformis, Cyperus compressus, Echinochloa colonum, Fimbristylis bisumbellata, Fimbristylis argentea, Panicum repens, Paspalum distichum, Rotala rotundifolia, Pistia stratiotes, Potamogeton crispus Sediment Retention Dense root network Fibrous roots fine root hairs Rapid root growth Root depth Rhizome formation Above ground biomass Alternanthera philoxeroides, Bacopa monnieri, Potamogeton crispus, Azolla pinnata, Lemna perpusilla, Pistia stratiotes, Nymphoides hydrophyllum, Trapa natans var. bispinosa, Pontederia crassipes, Spirodela polyrhiza, Hydrilla verticillate, Fimbristylis bisumbellata Water Quality Improvement Root exudates and high nutrient uptake Dense root network and pollutant absorption High biomass production and mycorrhizal associations Surface area for gas exchange Alternanthera philoxeroides, Hydrilla verticillate, Lemna perpusilla, Azolla pinnata, Spirodela polyrhiza, Nymphoides hydrophyllum, Pistia stratiotes, Pontederia crassipes, Potamogeton crispus, Trapa natans var. bispinosa, Bacopa monnieri, Rotala rotundifolia Fodder High biomass production Palatability for livestock High biomass Dense vegetation cover Alternanthera philoxeroides, Cyperus iria, Cyperus difformis, Cyperus compressus, Echinochloa colonum, Fimbristylis bisumbellata, Fimbristylis argentea, Panicum repens, Paspalum distichum Table 7. Traits and ecosystem services of key species. Tabela 7. Lastnosti in ekosistemske storitve ključnih vrst. 17 Acta Biologica Slovenica, 2025, 68 (1) (Lumpkin and Plucknett, 1982). These species significantly contribute to wetland biodiversity and ecological dynamics. The dominance of Cyperaceae (20.51%) is common in trop- ical and subtropical wetlands (Gopal, 2016). Sedge-domi- nated vegetation supports food webs, creates habitats, and removes toxic compounds from water. It provides food for insects, birds, and mammals, with some species utilized by humans (Mishra et al., 2016). Cyperaceae species are well-adapted to wetland conditions and play crucial roles in nutrient cycling and sediment stabilization. The signifi- cant presence of Poaceae (6.41%) further emphasizes the importance of graminoid species. The diversity of other dominant families (Araceae, Polygonaceae, Hydrocharita- ceae), including existing families suggests a complex wet- land flora supporting various ecological niches, potentially indicating overall health and resilience to environmental changes (Mitsch and Gosselink, 2015). Further research is needed to understand the specific ecological roles and interactions of these families within the studied wetlands. The species frequency analysis reveals distinct distri- bution patterns across the wetlands. High-frequency spe- cies like Pontederia crassipes, Alternanthera philoxeroides, and Hydrilla verticillata demonstrate adaptability to various conditions, with A. philoxeroides being a known invasive species (https://www.iucngisd.org/gisd/). The abundance of Nymphoides hydrophyllum and N. indica aligns with Preston and Croft's (2022) findings on species with effec- tive reproductive strategies. High frequencies of Pistia stratiotes and Potamogeton crispus may indicate nutrient enrichment (Guntenspergen et al., 2020), potentially due to wastewater inflow. Low-frequency species like Acmella paniculata and Torenia crustacea show localized distri- butions, suggesting specific habitat requirements or less competitiveness (Mishra et al., 2016). The restricted distri- bution of Bacopa monnieri and Persicaria glabra indicates potential habitat specificity or vulnerability to competition and disturbance. Urban wetlands face significant anthro- pogenic pressures affecting sensitive species (Grime, 2001). Azolla pinnata, Lemna perpusilla, and Spirodela polyrhiza, observed with low frequencies in W6, may be restricted due to environmental factors (Gopal, 2015). The species range reflects wetland ecosystem complexity, with high-frequency species shaping habitats and supporting ecosystem functions, while low-frequency species contrib- ute to biodiversity and resilience (Mishra et al., 2016). In comparison to Raunkaier's law of frequency classes (1934), all the wetlands (W1-W7) revealed heterogeneous vegetation structures, consistent with similar research conducted in the Hokersar wetland in the Kashmir Himalaya (Mir et al., 2009). W1 displayed an even species distribution, suggesting a more stable ecosystem likely resulting from balanced environmental conditions and minimal extreme disturbances. Similarly, sites W4 and W7 showed some degree of balance, contributing to ecological resilience. The importance of species distribution and evenness for ecosystem stability in wetlands is further emphasized by the work of Mälson et al. (2008). Their study on rich fen vegetation revealed that drainage significantly reduced biodiversity, while the slow recovery observed post-rewet- ting underscores the critical role of maintaining evenness for ecosystem resilience. Conversely, W2, W3, W5, and W6 exhibited complex patterns of species dominance and potential instability, with deviations from Raunkaier's normal frequency. Particularly, sites W2 and W5 exhibited notable species dominance, with W2 showing a higher frequency in class B than Raunkaier's normal frequency, while W5 displayed a decrease in class A, increases in classes B and C, and an absence of class E. These patterns suggest less stable ecosystems facing extreme conditions and competi- tive interactions (Tilman, 1982). Zedler and Kercher (2005) highlighted wetlands' vulnerability to invasions, noting that 24% of the world's most invasive plants are wetland species. They attributed this to wetlands acting as landscape sinks accumulating debris, sediments, water, and nutrients con- ditions that can facilitate invasions by creating canopy gaps or promoting opportunistic plant growth. Luo et al. (2010) demonstrated that intraspecific and interspecific competi- tion influence biomass accumulation nutrient accumulation in marsh plants, with competition strongest at lower water levels, while facilitation is more pronounced at higher water levels. The analysis reveals the complex interplay of envi- ronmental factors, competitive interactions, and ecosystem stability in these wetlands, emphasizing the utility of Raun- kaier's frequency classes in understanding wetland ecosys- tems and the importance of conservation efforts to maintain species evenness for long-term ecosystem stability. The analysis of four different biodiversity indices across seven wetlands (W1-W7) provides a comprehensive understanding of ecosystem health, species richness, and distribution patterns. The Shannon-Wiener diversity index (H') revealed varying levels of species complexity, with W7 exhibiting the highest biodiversity (3.4968) and W2 the lowest (2.9512). This pattern aligns with studies on the Han- jiang River wetlands, which demonstrated varied H' values 18 Acta Biologica Slovenica, 2025, 68 (1) based on plant community structure and human impact (Li et al., 2022). Simpson's index of diversity (1-D) corroborated these findings, showing high values (0.9505 to 0.9654) for W1, W3, W4, W6, and W7, indicating diverse and stable ecosystems. However, W2 and W5 displayed slightly lower (1-D) values (0.9367 and 0.9464, respectively), suggesting potential biodiversity reduction or species dominance, consistent with findings on the impact of urbanization in the Bamenda Municipality (Asongwe et al., 2022). The Mar- galef Diversity Index (DMg) provided insights into species richness, with W1, W3, W4, W6, and W7 showing relatively high values (3.887354 to 4.207085), indicating rich species diversity and favourable conditions for plant life. These high values likely reflect optimal environmental conditions and effective conservation efforts, aligning with the diverse and healthy ecosystems observed in Lake Alakol (Zaparina et al., 2024). Conversely, W2 (3.853248) and W5 (3.628268) exhibited lower DMg values, suggesting reduced species richness, possibly due to environmental stressors such as pollution, human activities, or invasive species (Saluja and Garg, 2017). These findings mirror challenges faced by ecosystems impacted by anthropogenic influences, similar to observations in the tropical lakes of the Central Ganga Plain (Dubey et al., 2022). Pielou's Evenness (J') analysis revealed high evenness values for W3, W4, W5, W6, and W7 (0.94020 to 0.96965), indicating stable environmental conditions with minimal species dominance. Interestingly, while W5 showed lower values in other indices, it main- tained high evenness, suggesting a relatively balanced dis- tribution of species despite potential reductions in overall diversity. W1 (0.92904) and W2 (0.91683) displayed lower evenness, aligning with their performance in other indices. These findings are consistent with Zhao et al. (2021), who reported that farmland abandonment positively influences evenness by transitioning plant communities from annual to perennial species. The lower values of W1 and W2, as noted by Sharma and Singh (2017), may reflect the effects of seasonal fluctuations and vegetation on evenness. The combined analysis of these four biodiversity indices paints a nuanced picture of the seven wetlands' ecological health. W7 consistently emerges as the most diverse and stable ecosystem, followed closely by W3, W4, and W6. These wetlands likely benefit from optimal environmental conditions and effective conservation efforts. W5 presents an interesting case, with lower diversity and richness but maintaining high evenness, suggesting a need for targeted conservation strategies to enhance its overall biodiversity while preserving its balanced species distribution. W1 and W2 consistently show signs of ecological stress across all indices, indicating they are prime candidates for intensive conservation and restoration efforts. These findings underscore the importance of comprehensive biodiversity assessments in wetland management and highlight the need for tailored conservation strategies that address the specific challenges faced by each wetland ecosystem. Long-term monitoring, such as that conducted in the Yellow River Delta since 2001, has shown that restored wetlands can significantly improve water quality and soil conditions, with vegetation quickly re-establishing and providing habi- tat for diverse bird species (Cui et al., 2009). Implementing such targeted conservation strategies could help improve diversity in degraded wetlands and ensure long-term eco- logical health across various wetland ecosystems. The Important Value Index (IVI) of each macrophyte of the studied wetlands reveals significant patterns in species composition and ecological importance (Mori et al., 2015). The findings indicate a clear stratification of species based on their IVI values: Low IVI species, such as Acmella panicu- lata and Hydrilla verticillata, exhibit limited ecological roles but contributed to overall biodiversity (Junk et al., 2013). The majority of species, comprising 58-77% across study sites (W1-W7), demonstrated intermediate IVI values. These species, including Ipomoea aquatica and Nymphoides indica, play crucial roles in maintaining ecological stability and highlight the complexity of wetland ecosystems. Of particular concern are the high IVI species, approxi- mately 21 in total, including Alternanthera philoxeroides, Pontederia crassipes, and Pistia stratiotes, which emerge as key invasive components of the wetland ecosystems (Zedler and Kercher, 2005). These dominant species, while indicative of successful adaptation to the wetland environment, raise concerns about their potential negative impacts on native biodiversity and ecosystem functions, necessitating careful monitoring and management strat- egies. The dominance of emergent species, followed by submersed, rooted floating-leaves, and free-floating growth forms, aligns with other wetland studies (Junk et al., 2013). This pattern suggests possible terrestrialization or eutrophication processes (Mitsch and Gosselink, 2015). While IVI provides valuable insights, it has limitations in capturing the functional roles of less abundant species (Frieswyk et al., 2007). The study emphasizes the need for balanced management strategies that consider both high and low IVI species to maintain biodiversity and ecosys- 19 Acta Biologica Slovenica, 2025, 68 (1) tem resilience (Zedler and Kercher, 2005). This analysis provides a comprehensive understanding of wetland plant community structure and species importance, highlighting the need for accurate, adaptive management approaches. Future research should focus on long-term monitoring and integration of functional trait analyses with IVI data to gain deeper insights into ecosystem processes and community assembly mechanisms in these wetland ecosystems. The identification of nine rare species is significant for conservation. Species like Grangea maderaspatana and Scleromitrion brachypodum, with higher IVI values among rare species, are of particular interest. Their rarity may be due to specific habitat requirements or environmental sensitivity (Grime, 2001). Rare submerged species such as Ottelia alismoides and Ceratopteris thalictroides are noteworthy, as they are often sensitive to water quality and habitat changes (Carpenter and Lodge, 1986). Their low abundance could indicate ecosystem stress, possibly from urbanization and pollution in wetlands W2 and W5. Elo et al. (2018) examined how environmental factors and human activities affect aquatic macrophyte diversity and rarity. The rarity of Pouzolzia zeylanica in W2, potentially due to habitat degradation and pollution, emphasizes the need for targeted conservation strategies. Salgado et al. (2023) found that protected land enhances native aquatic macrophyte survival and limits invasive species' spread. Monitoring and managing the impact of invasive species like Pontederia crassipes on rare species populations is crucial for maintaining ecological balance. The study has highlighted the crucial role of plant functional traits, particularly root traits, in delivering various ecosystem services (Faucon et al., 2017; Bardgett et al., 2014; Burylo et al., 2012). Root traits contribute significantly to various ecosystem functions. For instance, sediment retention is enhanced by dense root networks, fine root hairs, rhizome formation, rapid root growth, fibrous roots, and substantial aboveground biomass (Stokes et al., 2009; Borin et al., 2005; Blanco-Canqui et al., 2004). In terms of water quality improvement, macrophytes exhibit high nutri- ent uptake, pollutant absorption, root exudates, high bio- mass production, and mycorrhizal associations (Vymazal, 2007). Furthermore, erosion control and soil stabilization are facilitated by extensive root systems and high root hair density. Based on these findings, macrophytes such as Bacopa monnieri, Alternanthera philoxeroides, Cyperus spp., Echinochloa colonum, Fimbristylis spp., Panicum repens, Paspalum distichum, Rotala rotundifolia, Pistia stratiotes, and Pontederia crassipes exhibit traits such as extensive root systems and high root hair density. These traits enable these species to provide essential ecosystem services, including erosion control, soil stabilization, and nutrient absorption. In addition to these services, they also offer wildlife habitat, food, and fodder. Aquatic mac- rophytes play a crucial role in wastewater treatment and the management of algal blooms by providing essential ecosystem services. The provisioning services they offer include direct benefits such as nutrient removal and bio- mass production, which significantly improve water quality and create habitats for aquatic organisms. For example, Azolla pinnata excels in nutrient uptake and pollutant absorption (Rai, 2008), while Lemna perpusilla rapidly absorbs nutrients, effectively mitigating eutrophication (Vymazal, 2007). Additionally, regulating services are evident in how these species help to maintain ecological balance. Hydrilla verticillata, with its high root hair density that increases surface area, is particularly effective in treat- ing nutrient-rich wastewater (Dhir, 2013), and Pontederia crassipes contributes high biomass production that aids in pollutant absorption (Raven et al., 2005). Furthermore, Spirodela polyrhiza supports nutrient cycling and overall ecosystem health through its rapid nutrient uptake (Cronk and Fennessy, 2016). Collectively, these species not only enhance water quality but also foster biodiversity within aquatic ecosystems. Similarly, Potamogeton crispus, Hydrilla verticillata also provides habitat and food resources for various aquatic organisms. In terms of provisioning ser- vices, species like Azolla pinnata, Lemna perpusilla, Pistia stratiotes, and Spirodela polyrhiza are utilized as animal feed and biofertilizers, promoting agricultural sustainability. Furthermore, Nymphoides hydrophyllum, Marsilea minuta and Ipomoea aquatica serve as fodder for livestock, with M. minuta and Ipomoea aquatica also being consumed as a vegetable. Traditional medicinal practices utilize plants such as M. minuta and Bacopa monnieri for their thera- peutic properties. Additionally, Trapa natans var. bispinosa produces edible seeds that are valuable for both human diets and traditional medicine. Conclusion This pioneering study in the Eastern Ranchi District of Jharkhand, India, identified 78 macrophyte species across seven wetlands (W1-W7), revealing a predominance of 20 Acta Biologica Slovenica, 2025, 68 (1) emergent species (69%) and underscoring the richness of these ecosystems. The analysis documented nine rare species and 21 key species that contribute significantly to essential ecosystem services like soil stabilization and water quality improvement. High-frequency species such as Pontederia crassipes (85.71% in W1) and Hydrilla verticillata (57.14% in W1) demonstrated resilience across diverse habitats, while low-frequency species like Acmella paniculata (14.29% in W1) were limited to specific areas due to habitat sensitivity. Raunkiaer's frequency analysis indi- cated a dominance of Class B species in most wetlands, particularly W1 (64.52%), while W2 exhibited signs of dis- turbance with a higher proportion of Class A species (52%) and fewer in Classes C and E. Wetland W7 had the highest biodiversity (H' = 3.4968) and richness (DMg = 5.08069), while W2 had the lowest values, and Wetland W6 showed the highest evenness (J' = 0.96965), indicating ecological stability. Intermediate IVI species dominated most wetlands (58–77%), with Pontederia crassipes (IVI = 20.72) prominent in W1, and Acmella paniculata (IVI = 3.12) less so. This study provides essential baseline data for developing targeted conservation strategies to address ecological stress in urban wetlands (W1, W2, and W5). It emphasizes the crucial role of macrophytes in sustaining ecosystem services, highlighting their importance for wetland conservation and biodiversity management. Acknowledgements The authors are grateful to the local villagers, notably Samir Kumar of Bundu, Badan Dash of Rahe, Chamera Kuiri of Choga village, Niroj Kumar of Kita village, and Mishir Lal Rajak of Muri, for their persistent cooperation in supplying primary data during field surveys. Additionally, the authors express their heartfelt gratitude to Mr. Arup Kumar Chaud- hary, District Fishery Officer, Ranchi, Jharkhand, for kindly giving useful information about the wetlands of Eastern Ranchi. Funding This research received no external funding. 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The participants were 13–14-year-olds who had not yet learned about human evolution (the control group) and 15–16-year-olds who had learned about human evolution one year earlier in biology classes (the experimental group). The results show that students significantly improved their knowledge of human evolution with small to medium effect sizes. Students knew very well that modern humans are the only species of humans today and that they did not live at the same time as dinosaurs. The achievements were low on the following topics: modern humans lived at the same time as Neanderthals and mammoths, and modern humans did not evolve from Neanderthals. To the problematic topics, attention should be paid to teacher education and the biology curriculum. Few correlations were found between the knowledge of human evolution, acceptance of evolution, religiosity, and attitudes toward biology. Keywords knowledge of human evolution; acceptance of evolution; religiosity; attitudes toward biology; primary school 1 University of Ljubljana, Biotechnical Faculty * Corresponding author: E-mail address: jelka.strgar@bf.uni-lj.si Citation: Strgar, J., (2024). The effectiveness of teaching human evolution to 14- to 15-year-olds in Slovenia. Acta Biologica Slovenica 68 (1) Received: 15.05.2024 / Accepted: 07.10.2024 / Published: 28.10.2024 https://doi.org/10.14720/abs.68.01.18729 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license 24 Acta Biologica Slovenica, 2025, 68 (1) Učinkovitost poučevanja evolucije človeka pri 14-do 15-letnikih v Sloveniji Izvleček V Sloveniji se pri biologiji evolucijo človeka prvič učijo 14- do 15-letniki. Za mnoge od njih bo to v šoli edini stik s to temo. Cilj raziskave je bil ugotoviti, koliko znanja evolucije človeka so učenci usvojili. Podatke smo zbrali z vprašalnikom s petstopenjsko Likertovo lestvico. Udeleženci so bili 13- do 14-letniki, ki se še niso učili evolucije človeka (kontrolna skupina) in 15- do 16-letniki, ki so se evolucijo človeka učili pri biologiji eno leto pred tem (poskusna skupina). Resultati kažejo, da so učenci signifikantno izboljšali svoje znanje evolucije človeka, in sicer z majhno do srednjo velikostjo učinka. Učenci so dobro vedeli, da je moderni človek danes edina vrsta človeka in da ni živel sočasno z dinozavri. Dosežki pa so bili nizki pri naslednjih temah: moderni človek je živel sočasno z neandertalcem in mamutom, moderni človek se ni razvil iz neandertalca. Problematičnim temam je treba posvetiti pozornost v izobraževanju učiteljev in osnovnošolskem učnem načrtu. Med znanjem evolucije človeka, sprejemanjem evolucije, vernostjo in odnososm do biologije je bilo malo korelacij. Ključne besede znanje evolucije človeka; sprejemanje evolucije; vernost; odnos do biologije; osnovna šola Introduction Human evolution is a topic that students can quickly lose interest in, especially if the teacher distracts them with too much theory and data that make no sense to them (Flammer, 2006). Motivation to learn about evolution is influenced by many factors, one of which is prior knowledge. Bloom and Weisberg (2007) claim that students' prior knowledge is the main reason for not accepting evolution, as misconceptions and beliefs can hinder the process of accepting new knowl- edge (Wescott & Cunningham, 2005; Smith, 2010; Yates & Marek, 2014). If the prior knowledge is good, the student can add new knowledge to the already existing one. However, if the student has misconceptions, motivation decreases, as it is extremely difficult to change entrenched misconceptions with classical teaching methods. Slovenia ranks above average in the PISA survey. In the latest survey, which was conducted in 2022, Slovenian 15-year-olds ranked above average in scientific literacy. From these results, we can conclude that science literacy among primary and secondary school students in Slovenia is good, and it is also stable according to prior PISA surveys (Strgar, 2010; Štraus et al., 2016). In a survey about the acceptance of the theory of evolution (Miller et al., 2006), Slovenia ranked in 16th place among 34 countries. University students in Slovenia think that understanding evolutionary concepts is a necessary part of solid general education, but there is also evidence that their actual understanding of evolution is not satisfac- tory. Surprisingly, they also think that alternative nonscien- tific theories of evolution should be presented in schools (Šorgo et al., 2014). According to Pinxten et al. (2020), most of the popula- tion in many countries has a very limited formal introduction to the understanding of evolutionary theory. However, in most countries, there are national regulations that demand scientific teaching of evolutionary theory (Borgerding et al., 2016; Tavares & Bobrowski, 2018). In Slovenia, the theory of evolution was first introduced in school in 1945 (Vesel, 1977). According to the current national curriculum (Učni načrt. Program osnovna šola. Biologija, 2011), 14- to 15-year-old students learn about biological evolution in biology classes for the first time. However, some evolutionary concepts are already included in the curriculum for history that is taught to younger students (Učni načrt. Program osnovna šola. Zgodovina, 2011). At the age of 14 to 15, students in Slovenia finish their compulsory primary education. Biological evolution is introduced to them in the final year (Učni načrt. Program osnovna šola. Biologija, 2011). There is one single national curriculum for all students up to this age. Afterwards, the students attend different secondary schools according to their career choices. These schools vary in the amount of biology courses that they offer. This means that some 25 Acta Biologica Slovenica, 2025, 68 (1) of the students continue their education at secondary schools, which have no evolution as part of their curricu- lum. Into their adult life, these students take with them only the knowledge of evolution they had learned in primary school. Therefore, it is important to know what level of understanding of evolution the Slovenian school system provides during obligatory primary education. In the Slovenian national curriculum for 14 to 15-year- olds, there are 18 objectives concerning evolution and only one objective regarding human evolution, namely, “Students can explain the origin of humans and primates and the kinship of humans with other primates” (Učni načrt. Program osnovna šola. Biologija, 2011, p. 17). Published research shows that understanding evolu- tion is challenging for students at all levels of education for a variety of reasons. Surroundings and socio-economic factors also influence students and take part in structur- ing students’ preconceptions about evolution and their attitudes toward evolution (da Silva Oliveira et al., 2022). The problem, therefore, is how much students can learn at that age, specifically, how well they can understand evolutionary concepts and what contextual factors shape students’ knowledge. Many studies (Miller et al., 2006; Hermann, 2013; Mantelas & Mavrikaki, 2020; da Silva et al., 2022; Salazar-Enriquez et al., 2023) confirmed a negative correlation between acceptance of evolution and religiosity. It differed depending on the country and cultural background of the people tested. For Slovenia, this prob- lem was addressed in studies by Šorgo et al. (2014), Kralj et al. (2018), and Torkar and Šorgo (2020). This research aimed to establish how much knowledge about human evolution students had before they learned about this topic in formal education and how much new knowledge about human evolution they acquired during formal education. Therefore, the aim was to determine how effective were the lessons on human evolution, which, according to the national curriculum for biology (Učni načrt. Program osnovna šola. Biologija, 2011), were attended by students aged 14 to 15 years. The point of interest was also whether contextual factors, such as attitudes toward biology, acceptance of evolution, and religiosity, were con- nected with students’ knowledge of human evolution. The outcome could help make changes in the current national curriculum as well as be guidance for teacher educators and teachers themselves (Salazar-Enriquez et al., 2023). The following research questions were formulated: RQ1: How effective are lessons about human evolution in Slovenia taught to 14–15-year-old students? RQ2: Are the chosen contextual factors (students’ accep- tance of evolution, attitudes toward biology, and religiosity) connected with students' achievements on the knowledge test about human evolution? Materials and Methods General Background The data were gathered in the 2021/22 and 2022/23 school years. The survey was anonymous; all the parents of the students signed an agreement to allow their children to participate. No benefits were offered to the participants. According to Slovenian legislation, such a survey does not need the approval of an ethics committee or similar body. Sample Our sample consisted of 593 students. The national curric- ulum for biology in Slovenia (Učni načrt. Program osnovna šola. Biologija, 2011) mandates for evolution to be taught to 14- to 15-year-olds. Students of this age were not part of the sample. In the survey, there were one year younger students (13- to 14-year-olds; a control group; n = 364).) who had not yet learned about human evolution. The experi- mental group (15- to 16-year-olds; n = 229) was represented by the students who had learned about human evolution in biology classes in the previous school year. In the sample, there was a slightly higher percentage (53.5%) of females compared to males. Instrument and Procedures Data were collected using a questionnaire. In the begin- ning, there were two demographic questions (gender and grade). These were followed by 59 statements that assessed students’ knowledge of human evolution, their acceptance of evolution, attitudes toward biology, and religiosity. This part of the questionnaire used a 5-point Likert scale, ranging from strongly disagree (1) to strongly agree (5). Eleven items were originally worded in such a way that disagreement was required as a correct answer; these items were re-coded for statistical analysis. The questionnaire was compiled from statements used in sev- eral prior studies. The religiosity of participants was tested 26 Acta Biologica Slovenica, 2025, 68 (1) with ten statements using the PERF scale, which was created by Beniermann (2019) and translated into the Slo- venian language and used by Torkar and Šorgo (2020) on the Slovenian population. Eight statements that explored students’ attitudes toward biology were adapted from The Relevance of Science Education study (Sjøberg & Sch- reiner, 2019). Seventeen statements that tested students’ acceptance of evolution were taken from the MATE ques- tionnaire (Rutledge & Sadler, 2007), and ten were adapted from the questionnaire used by Miller et al. (2006). The remaining 14 statements that tested students’ knowledge about human evolution were partly adapted from the study by Miller et al. (2006) and partly designed in line with the curriculum and the textbooks that the participants of this study used when learning about evolution. Face validity of these 14 statements was ensured by the author and the three biology teachers who judged and revised them. The questionnaire was administered during students’ regular biology classes. Students completed it in approxi- mately 15 minutes. Data analysis Data were tested for normal distribution with the Kolmog- orov-Smirnov test; because the distribution of data was not normal (ps < 0.001), non-parametric testing was used. The statistical significance of differences in knowledge about human evolution between the control and the experimental groups was established by a Mann-Whitney U test. Effect size r was calculated using the equation r = Z/√N. On 14 statements of the knowledge test about human evolution, descriptive statistics (mean, standard deviation) were used. On 45 statements assessing acceptance of evolution, attitudes toward biology, and religiosity, principal component analysis (PCA) was conducted using the orthogonal rotation (varimax) method with Kaiser nor- malization (Table S1). The value of the Kaiser-Meyer-Olkin measure of sampling adequacy was .925, which means that the sample size was adequate for PCA. Bartlett’s test of sphericity was highly significant (χ2 = 11253.468, df = 990, p < .001), indicating that correlations between statements were sufficiently large for PCA. According to the scree plot, retaining five components was justified. Following this, three statements were removed from the analysis. Thirteen statements loaded onto component 1 (religiosity); 14 state- ments loaded onto component 2 (acceptance of evolution – the theory); eight statements loaded onto component 3 (attitudes toward biology); four statements loaded onto component 4 (acceptance of evolution – the theory is (not) scientific), and three statements loaded onto component 5 (acceptance of evolution – organisms do (not) evolve). Cronbach’s alphas for three components were highly reli- able (from .85 to .91), while for two components, they were relatively less reliable (.53 and .68). These five components explained 55.926% of the variance. Cronbach’s alpha for all 42 statements was .83. Additional results are available as supplementary material accompanying the online article. Correlations between the knowledge of human evolution, acceptance of evolution, religiosity, and atti- tudes toward biology were analyzed using Spearman’s correlation coefficient (rs). Comparisons of these correla- tions were then calculated to establish if the differences between correlations of the two students’ age groups were statistically significant. For this, the following equation was used: Zdifference = (zr1-zr2)/√{[1/(N1-3)]+[1/(N2+3)]} (Field, 2009, p. 191). Results Students’ Achievements on the Knowledge Test about Human Evolution Students’ knowledge of human evolution was tested with 14 statements. Results show that all students showed the least knowledge (M = 2.41-2.57) in statements rE1 (Modern humans did not evolve from Neanderthals.), E2 (Modern humans lived in today's Slovenia at the same time as mammoths), and E36 (Modern humans and Neanderthals lived on Earth at the same time) (Tab. 1). In nine statements, they showed an average knowledge (M = 3.03-3.43). The highest knowledge (M = 3.63-4.13) was achieved on the following two statements: E3 (Today, all human species are extinct, except for our species, modern humans), rE38 (Modern humans did not live in today's Slovenia at the same time as dinosaurs.). Differences among Students’ Achievements on the Knowledge Test about Human Evolution by Age On 12 out of 14 statements about the knowledge of human evolution, 13-14-year-olds answered significantly differently than 15-16-year-olds (Tab. 1). On one of those 12 27 Acta Biologica Slovenica, 2025, 68 (1) statements (rE33 Modern humans did not evolve less than 15,000 years ago.), 13–14-year-olds showed more knowl- edge. On the remaining 11 statements, 15–16-year-olds had more knowledge. The effect sizes of differences in knowledge between 13–14-year-olds and 15–16-year-olds were mostly small (on 8 out of 14 statements) to medium (6 statements). Correlations between Students’ Achievements on the Knowledge Test about Human Evolution and Contextual Factors Correlations for the subsample of 13–14-year-olds and the subsample of 15–16-year-olds were established, respec- tively. The variables were the average achievement on Age (years) All students (N = 589) 13-14 (n = 360) 15-16 (n = 229) Mann-Whitney U test Effect Size Statement M SD M SD M SD U Z p r rE1 Modern humans did not evolve from Neanderthals.* 2.41 1.229 2.29 1.183 2.60 1.276 35473.500 -2.742 .006 -.11 E2 Modern humans lived in today's Slovenia at the same time as mammoths. 2.51 1.237 2.44 1.248 2.62 1.215 36871.000 1.780 .075 .07 E36 Modern humans and Neanderthals lived on Earth at the same time. 2.57 1.348 2.43 1.267 2.79 1.441 33830.000 -2.933 .003 -.12 rE33 Modern humans did not evolve less than 15,000 years ago.* 2.99 1.045 3.06 1.060 2.89 1.015 36477.000 -2.108 .035 -.09 E34 Modern humans evolved in Africa. 3.03 1.086 2.78 1.084 3.43 .964 27174.500 -7.220 <.001 -.30 rE35 Humans did not evolve from chimpanzees. 3.08 1.183 2.81 1.102 3.49 1.185 28028.000 -6.466 <.001 -.27 E4 Neanderthals and modern humans share 99.7% of their genes. 3.14 .967 3.03 .924 3.32 1.008 33892.500 -3.532 <.001 -.15 E19 Neanderthals lived in Europe, Asia, and the Middle East. 3.30 .921 3.22 .962 3.41 .843 35868.500 -2.478 .013 -.10 E37 Humans's three closest relatives in terms of evolutionary development are gorillas, orangutans, and chimpanzees. 3.38 1.073 3.36 1.092 3.42 1.043 38414.500 -.774 .439 -.03 rE26 Humans today do not have more or less the same shape as we have always had.* 3.40 1.052 3.21 1.016 3.70 1.040 30096.500 -5.518 <.001 -.23 E41 Humans and chimpanzees evolved from a common ancestor. 3.42 1.089 3.22 1.060 3.74 1.062 29122.000 -6.138 <.001 -.25 rE5 The common ancestor of humans and chimpanzees does not live in Africa anymore.* 3.43 1.107 3.34 1.093 3.57 1.116 35846.000 -2.333 .020 -.10 E3 Today, all human species are extinct, except for our species, modern humans. 3.63 1.198 3.41 1.179 3.99 1.141 28594.500 -6.177 <.001 -.26 rE38 Modern humans did not live in today's Slovenia at the same time as dinosaurs.* 4.13 1.174 3.86 1.282 4.54 .823 28347.500 -6.678 <.001 -.28 Table 1. Differences in the students’ knowledge of human evolution and effect sizes of differences. Tabela 1. Razlike v znanju učencev o evoluciji človeka in velikost učinka razlik. *reversed statement 28 Acta Biologica Slovenica, 2025, 68 (1) the knowledge test about human evolution and five com- ponents extracted by PCA: religiosity (F1), attitudes toward biology (F3), acceptance of evolution – the theory (F2), acceptance of evolution – the theory is (not) scientific (F4), and acceptance of evolution - organisms do (not) evolve (F5). There were six significant correlations altogether (Tab. 2, Tab. 3). Acceptance of evolution – the theory is (not) sci- entific (F4) was significantly correlated with religiosity (F1) in both subsamples and also with acceptance of evolution – the theory (F2) in 15–16-year-olds. Acceptance of evolution - organisms do (not) evolve (F5) was significantly correlated with attitudes toward biology (F3) in 15–16-year-olds. Knowledge of human evolution was significantly correlated with acceptance of evolution – the theory (F2) and atti- tudes toward biology (F3) in the group of 13–14-year-olds. All correlations were small (.13 < rs < .21). Comparison of Correlations between the Control Group and the Experimental Group A comparison of correlations between the control and the experimental group of students was calculated. The correlations between the knowledge of human evolution and the five components obtained by PCA in the group that had not yet learned about evolution (13–14-year-olds) were mostly significantly different (ps < .01) from the correlations in the group that had already learned about human evolu- tion (15–16-year-olds). Only the correlation between accep- tance of evolution (i.e., the theory is (not) scientific (F4) and acceptance of evolution - organisms do (not) evolve (F5)) was similar in both age groups (p = .223). Discussion The first research question dealt with whether teaching about human evolution to 14–15-year-old students in Slo- venia is effective and whether it attains the standards set in the national curriculum for biology. Overall, the average achievements on the knowledge test of human evolution ranged from very poor (M = 2.41) to very good (M = 4.31). In summary, Slovenian students knew very well that modern humans did not live at the same time as dinosaurs (rE38) and that modern humans are the Name of the Wetlands, (Blocks). F1 F2 F3 F4 F5 F1 Religiosity 1.00 F2 Acceptance of evolution – the theory .06 1.00 F3 Attitudes toward biology .07 .00 1.00 F4 Acceptance of evolution - the theory is (not) scientific .16* -.07 -.00 1.00 F5 Acceptance of evolution - organisms do (not) evolve .12 -.05 -.13 -.05 1.00 Knowledge of human evolution -.08 .13* -.18** -.00 .03 Name of the Wetlands, (Blocks). F1 F2 F3 F4 F5 F1 Religiosity 1.00 F2 Acceptance of evolution – the theory .04 1.00 F3 Attitudes toward biology -.05 -.07 1.00 F4 Acceptance of evolution - the theory is (not) scientific -.15* .21** .07 1.00 F5 Acceptance of evolution - organisms do (not) evolve .04 -.04 .20** -.07 1.00 Knowledge of human evolution -.07 -.04 .09 -.02 .02 Table 2. Correlations between Students’ Achievements on the Knowledge Test about Human Evolution and Contextual Factors (13–14-year-olds). Tabela 2. Korelacije med dosežki učencev na testu znanja o evoluciji človeka in ozadenjskimi dejavniki (13-do 14-letniki). Table 3. Correlations between Students’ Achievements on the Knowledge Test about Human Evolution and Contextual Factors (15–16-year-olds). Tabela 3. Korelacije med dosežki učencev na testu znanja o evoluciji človeka in ozadenjskimi dejavniki (15-do 16-letniki). * p < .05, ** p < .01 * p < .05, ** p < .01 29 Acta Biologica Slovenica, 2025, 68 (1) only species of humans today (E3). They also knew above averagely well that humans had changed their shape over time (rE26), that humans and chimpanzees evolved from a common ancestor (E41) that no longer lives (rE5), and that gorillas, orangutans and chimpanzees are humans' closest relatives (E37). Students knew averagely well that modern humans evolved in Africa (E34), that they evolved more than 15,000 years ago (rE33), and that humans did not evolve from chimpanzees (rE35). In two of these statements, the knowledge improved significantly after the biology lessons in the ninth grade. Students knew beyond average that modern humans and Neanderthals lived at the same time (E36), that modern humans lived at the same time as mammoths (E2), and that modern humans did not evolve from Neanderthals (rE1). For two of these statements, the biology lesson slightly improved knowledge. In contrast, students were unsure what proportion of genes Neanderthals and modern humans share (E4) and where Neanderthals lived (E19), although knowledge improved significantly after the biology lessons in the ninth grade of primary school. The fact that even the younger students had above-av- erage achievements in seven out of 14 statements suggested that they might have gained some of this knowledge in history classes one year earlier (Učni načrt. Program osnovna šola. Zgodovina, 2011), through popular media (Modell & Wenderoth, 2005), digital and print media (Garibi et al., 2021), or their social surroundings (OECD, 2016; OECD, 2017; Šterman Ivančič & Mlekuž, 2023), espe- cially their parents (Salazar-Enriquez et al., 2023). The comparison of the knowledge of both groups showed that there were significant differences between stu- dents' answers in most of the statements. The experimental group (15–16-year-olds) showed mostly a better knowledge of human evolution than the control group (13–14-year- olds). Interestingly, younger students showed significantly better knowledge of one statement (rE33, Modern humans did not evolve less than 15,000 years ago). No knowledge was gained on the following two statements (E37) Human three closest relatives in terms of evolutionary develop- ment are gorillas, orangutans, and chimpanzees, and (E2) modern humans lived in today's Slovenia at the same time as mammoths. This suggested that these three topics were probably not addressed in biology classes. On the remain- ing 11 statements, older students answered significantly better. Therefore, we can conclude that in Slovenia, the teaching of (at least human) evolution at the primary school level is reasonably good. This is in line with Mead et al. (2017), who found that after classes, students' knowledge of evolution was improved immediately after the lesson and was retained. Some studies in other European coun- tries showed that teaching evolution was not satisfactory and that evolution was taught only after the other topics had been covered (Pinxten et al., 2020). Some teachers also do not have sufficient content knowledge (Borgerding et al., 2015; Hartelt et al., 2022) and pedagogical skills to teach such a demanding topic (Stasinakis & Athanasiou, 2016). However, the fact that the effect sizes of all eleven improvements in knowledge of human evolution in the present study were mostly small or medium suggested that there is room for improvement in the way human evolution is taught. Changes in the curriculum should be made con- cerning the topics with low achievements. Despite some research not showing any connection between students' knowledge and the quality of the national curriculum (Belin & Kisida, 2015), other studies showed that the structure of the curriculum influences knowledge and acceptance of evolution (Vaughn & Robbins, 2017). Information gathered in the present study can be applied immediately to improve the curriculum (Sala- zar-Enriquez et al., 2023), which is currently underway in Slovenia. The results will also be important for pre-service teachers' educational programs because by securing the understanding and acceptance of evolution in teachers, the transfer of these to new generations of students will be more favourable (Balgopal, 2014; Cofre et al., 2017; Torkar & Šorgo, 2020). The second research question dealt with the chosen contextual factors (students' religiosity, attitudes toward biology, and acceptance of evolution) and their possible correlations with students' achievements on the knowl- edge test about human evolution. Six significant but small correlations were found. According to most studies, the knowledge and accep- tance of evolution are positively correlated (Carter et al., 2015; Tavares & Bobrowski, 2018; Torkar & Šorgo, 2020; Salazar-Enriquez et al., 2023). Surprisingly, there was no such correlation for 15–16-year-olds in the present study. However, for 13–14-year-olds, the results showed that the better knowledge they possessed, the more they accepted the general statements of the evolutionary theory (F2). This is in line with other studies indicating that the correlation 30 Acta Biologica Slovenica, 2025, 68 (1) between knowledge and acceptance of evolution is not strong (Mead et al., 2017). Most of the research (e.g., Salazar-Enriquez et al., 2023) found that knowledge and acceptance of evolution negatively correlate with religious beliefs. Interestingly, the present study showed no correlation between religiosity and knowledge. However, in both age groups, there was a small negative correlation between religiosity and con- viction that evolutionary theory is scientific (F4). The more religious 15–16-year-olds were, the less convinced they were that the evolutionary theory is scientific. At the same time, more religious 13-14-year-old students were also more convinced that the evolutionary theory was scientific. This result is consistent with Mpeta et al. (2015) and Fiedler et al. (2024), who stated that the acceptance of evolution is also related to age, religiosity, and the time that has passed since the first learning about evolution. A small negative correlation was found between atti- tudes toward biology (F3) in 13- to 14-year-olds and knowl- edge of human evolution. This is not surprising—despite a positive attitude toward biology, these students lacked knowledge of human evolution because they had not been formally taught this topic yet. Students 15 to 16 years old with more positive attitudes toward biology (F3) were more convinced that organisms evolved (F5). This could be explained by the findings of Prokop et al. (2007) and Kubiatko et al. (2017), who report that good teaching strate- gies and a positive teacher personality positively influence students’ attitudes toward biology. It might be that students who were taught by such teachers were also more likely to accept the scientific facts presented by these teachers. The only correlation between two aspects of the acceptance of evolution was observed in 15- to 16-year-olds: the more the students accepted the theory of evolution (F2), the more they were convinced that it is scientific (F4). Correlations calculated for the 13- to 14-year-olds were then compared with the correlations calculated for the 15- to 16-year-olds for statistical significance. Fourteen out of 15 correlations calculated differed significantly between these two groups (ps < .01). The results show that teaching evolution not only significantly improved the students’ knowledge of human evolution but also caused changes in the acceptance of the evolutionary theory. This is in line with other studies that found positive correlations between knowledge and acceptance of evolution (Carter et al., 2015; Tavares & Bobrowski, 2018; Torkar & Šorgo, 2020; Salazar-Enriquez et al., 2023). There was no difference (p = .223) only regarding one correlation: in both groups, stu- dents who were more convinced that evolutionary theory is scientific (F4) were also more convinced that organisms evolve (F5). The fact that the 13- to 14-year-olds who had not learned evolution yet had the same convictions as the 15- to 16-year-olds was surprising, and it could be the con- sequence of contextual factors, such as the students’ social surroundings (OECD, 2016; OECD, 2017; Šterman Ivančič & Mlekuž, 2023) or parental education level (Salazar-En- riquez et al., 2023). A limitation of this study was its cross-sectional nature. A longitudinal study would have yielded more reliable results, but it would have been impossible to conduct due to logistical issues. Conclusions We can conclude that students in the ninth grade of ele- mentary school improved their knowledge of human evo- lution. The progress was small to medium. The question is how to eliminate some important misconceptions our students have concerning Neanderthals, specifically that modern humans evolved from Neanderthals (rE1) and that modern humans and Neanderthals did not live on Earth at the same time (E36). The student's knowledge should also be improved on the following topics: modern humans evolved in Africa (E34) more than 15,000 years ago (rE33), they did not evolve from chimpanzees (rE35), and they lived at the same time as mammoths (E2). In these topics, attention should be given to teacher education programs in curricular renewal. Author Contributions Conceptualization, J.S.; methodology J.S.; formal analysis, J.S.; investigation, J.S.; data curation, J.S.; writing—original draft preparation, J.S.; writing—review, editing, and paper communication, J.S. All authors have read and agreed to the final version of the manuscript. 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Gas chromatography was used to analyze the phytochemical profile. The docking tool, Cb-dock2, was used for in-silico analysis. G+ and G- bacterial strains were utilized to confirm the wet lab results. Chemical profiling revealed 18 peaks, and some major bioactive compounds were like phytol, 4-methyl-2- phenylindole and α-tocospiro A. Docking analysis revealed strong interaction against bacterial Penicillin-binding Protein (PBP1a) with docking scores ranging from -5.2 to -7.9 (Vina score). The PBP-Ligand complex has Van der Waals, hydrogen bond, pi-cation, and alkyl bonding interactions, according to the results of the interaction investigations. Calotropis latex successfully suppressed the development of bacterial strains (G+ and G-) with inhibition zones ranging from 1.1 cm to 2.2 cm, as demonstrated by in-vitro tests, indicating their potential as antibacterial medications. The study found that latex-based bioactive compounds have the potential to be used in the pharmaceutical and biotechnology sectors by facilitating the creation of structural alterations that lead to stronger medications. Therefore, this latex- derived compound may open a new pipeline into the development of multi- target antibiotics against a broad-spectrum multidrug-resistant G- bacterium. Keywords Penicillin-binding protein, calotropis latex, in-silico studies, anti-bacterial 1 P.G Dept of Biotechnology, Lyallpur Khalsa College, Mohyal Nagar Jalandhar, Punjab (144008), India * Corresponding author: E-mail address: arundevsharma47@gmail.com Citation: Sharma, A. D., Chauhan, A., (2024). Unveiling the antibacterial potential of latex- derived phytocomponents from Calotropis gigantea targeting bacterial Penicillin-binding proteins: chemical profiling, in silico docking, and in vitro lab validation. Acta Biologica Slovenica 68 (1) Received: 12.09.2024 / Accepted: 21.10.2024 / Published: 29.10.2024 https://doi.org/10.14720/abs.68.01.19803 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license 34 Acta Biologica Slovenica, 2025, 68 (1) Razkritje antibakterijskega potenciala iz lateksa pridobljenih fitokomponent iz Calotropis gigantea, usmerjenih proti bakterijskim proteinom, ki vežejo penicilin: kemijsko profiliranje, in silico docking in in vitro potrditev v laboratoriju Izvleček Nenadzorovana uporaba trenutno dostopnih zdravil je povzročila razvoj odpornosti bakterij proti antibiotikom po vsem svetu. Namen tega dela je bil izvesti analizo molekularnega prileganja in kemijsko profiliranje bioaktivnih sestavin, pridobljenih iz lateksa Calotropis gigantea, čemur bo sledila potrditev z eksperimenti. Za analizo fitokemičnega profila je bila uporabljena plinska kromatografija. Za in silico analizo je bilo uporabljeno orodje Cb- dock2. Za potrditev rezultatov v laboratoriju so bili uporabljeni sevi G+ in G- bakterij. Kemijsko profiliranje je pokazalo 18 vrhov, nekatere glavne bioaktivne spojine pa so bile fitol, 4-metil-2- fenilindol in α-tokospiro A. Analiza prileganja je pokazala močno interakcijo z bakterijskim proteinom, ki veže penicilin (PBP1a), z rezultati dokiranja od -5,2 do -7,9 (ocena Vina). V kompleksu PBP-ligand so glede na rezultate preiskav interakcij prisotne Van der Waalsove vezi, vodikova vez, pi-kation in alkilna vez. Lateks kalotropisa je uspešno zaviral razvoj bakterijskih sevov (G+ in G-) z inhibicijskimi conami od 1,1 cm do 2,2 cm, kot so pokazali in vitro testi, kar kaže na njihov potencialno uporabnost kot antibakterijska zdravila. Študija je pokazala, da imajo bioaktivne spojine na iz lateksa Calotropis gigantea potencial za uporabo v farmacevtskem in biotehnološkem sektorju, saj omogočajo ustvarjanje strukturnih sprememb, ki vodijo k močnejšim zdravilom, zato lahko ta spojina, pridobljena iz lateksa, odpre novo pot za razvoj multitarčnih antibiotikov proti širokemu spektru G- bakterij z multiplo rezistenco. Ključne besede penicillin vezajoč protein, mleček kalotropisa, in silico študije, protibakterijsko delovanje Introduction Since they were developed, antibiotics have significantly benefited human health and the battle against bacterial illnesses. However, in recent years, antibiotic abuse has resulted in medicine-resistance-created antibiotic-resistant microorganisms (ARMs), which present serious health risks to individuals. Therefore, the researchers' current goal is to develop innovative, safe drugs (Mir et al., 2022). The emergence of antibiotic resistance has made microbes one of the greatest risks to human health (Harris et al., 2022). G- bacteria are unique to hospitals and communities; they have developed resistance to all currently available antibiotics. β-lactam antibiotics have been targeting mem- brane-bound macromolecules involved in forming cell walls, known as penicillin-binding proteins (PBPs), for over 70 years. Numerous PBPs are found in bacteria, and some are starting to show their special roles in the cell division cycle (Straume et al., 2020). Peptidoglycan is depleted and may cause cell death when β-lactam antibiotics block tran- speptidation or carboxypeptidation activities (Chan et al., 2016). This powerful mechanism, which has made penicillin and its analogues the most extensively used antibiotics globally for all infectious illnesses over the past 70 years, is under pressure due to the emergence of penicillin and its analogue resistance. This highlights the necessity for cutting-edge, all-natural antibiotic remedies. Additionally, it has been observed that the naturally occurring substance moenomycin inhibits the actions of glycosyltransferases, which cause bacterial cells to die by breaking down pep- tidoglycan (Masters et al., 2020). Glycan chain polymer- ization (transglycosylation) and glycan chain cross-linking (transpeptidation) are catalyzed by PBPs. Certain PBPs hydrolyze the last D-alanine of the stem pentapeptide (DD-carboxypeptidation) or the peptide link between the two glycan chains (endopeptidation), according to Straume et al. (2020). One of the most promising antibiotic targets is the mechanism involved in bacterial cell wall production. A comparable macromolecular framework is absent from human metabolism, but many proteins, including PBPs, are 35 Acta Biologica Slovenica, 2025, 68 (1) involved in the metabolism of bacteria, which is essential to life. These two prerequisites must be met in order to pro- duce antibiotics. Furthermore, it has been noted that the naturally occurring compound moenomycin suppresses the activities of glycosyltransferases, which degrade peptidoglycan and kill bacterial cells (Masters et al., 2020). PBPs are thought to be great candidates for the creation of bacterial inhibitors due to their important role. The function of the PBP protein can be blocked to prevent bacterial replication. No PBP with a similar cleavage specificity has been found in humans; hence, the inhibitors are unlikely to be considered dangerous. Because they are safer, more ecologically friendly, and have no unfavourable side effects, conventional medical procedures are therefore improving and growing in pop- ularity (Mir et al., 2022). People still favour natural herbal remedies over synthetic ones despite the many advan- tages that synthetic drugs provide. Because they include multiple essential botanical components in various plant sections, medicinal plants are unique in that they may treat and cure a wide range of human health concerns (Salm et al., 2023; Chaachouay and Zidane, 2024). Natural plant products, like plant-based latex, are rich in bioactive com- pounds that vary in both biology and molecular structure, making them important tools for the creation of new drugs (Nasim et al., 2022). Calotropis gigantea is commonly known as the giant milkweed and belongs to the Asclepiadaceae family (Wijeweera et al., 2020). In traditional Ayurvedic medicine, Calotropis gigantea is called "Sweta Arka" and "Rakha Arka", respectively, and it is designated an ethnomedical useful plant (Julius et al., 2021). It is a perennial shrub native to diverse regions encompassing tropical Africa and Asia (Wijeweera et al., 2020). This species is widely distributed across roadsides, railway tracks and deserted land pockets and can be found in countries such as Bangladesh, Burma, China, India, Indonesia, Malaysia, Pakistan, the Philippines, Thailand, Nepal, and Sri Lanka, where it holds significant economic importance (Gyawali et al., 2020). Calotropis gigantea is commonly known as milkweeds because of the latex they produce (Wijeweera et al., 2020). Plants produce white latex when sliced or broken. Several plant parts, including the flowers, stem, and leaves, can be used to extract latex (Timilsina et al., 2020). This white latex is loaded with several bioactive compounds. However, due to the biologically active substances such as alkaloids, flavonoids, terpenoids, glycosides, phenolics, tannins, and saponins that it contains (Kumar et al., 2021), plant latex has made a significant contribution to medicine. Moreover, some ethnomedicinal applications of Calotropis plant parts include pesticide, anti-inflammatory, anti-fertility, antiulcer, fungicide, antiseptic, insecticide, anti-diabetic, and anti-hel- minthic activity (Wadhwani et al., 2021). Nevertheless, very little research on antioxidant bioactive compounds has been done on the latex of such a neglected plant species, even though this plant is widely utilized by the local com- munities as food ethnomedicine and is thought to be very nutritious. Consequently, in order to support Calotropis gigantea applications in traditional medicine, research into the latex secondary metabolite compositions, particularly its richness in phytonutrients, is still needed. Through the use of computational approaches to drug discovery, sophis- ticated methodologies that may be applied to screen drugs derived from phytochemicals found in a variety of medicinal plants have been established. Computational prediction models are critical to guide the technique selection pro- cess for drug and technology development. Furthermore, they have been used in the in-silico prediction of pharma- cokinetic, pharmacological, and toxicological parameters, per Loza-Mejía et al. (2018). Molecular docking has become a practical and affordable approach for drug discovery and evaluation. As per the findings of Lee et al. (2019), this tech- nique provides a valuable understanding of drug-receptor interactions and may be applied to predict the orientation of a potential medication upon binding to a target protein. However, because of its intricacy, the precise mechanism behind Calotropis gigantea latex's antibacterial action is ultimately unclear. We've spoken about how we think Calot- ropis gigantea latex's several bioactive ingredients may help reduce the threat posed by both G+ and G- bacteria that are resistant to multiple drugs. Consequently, further research is needed to determine the possible medicinal advantages and workable modes of action of Calotropis gigantea latex. The objective of this study was to perform phytochemical screening and in-silico docking of the most common compounds present in Calotropis gigantea latex against target proteins involved in the bacterial life cycle. Additionally, it will offer new insights into future projections for figuring out the dose of crucial antibacterial drugs. This will increase our knowledge of the potential therapeutic use of the latex from the Calotropis gigantea plant. These techniques seek to offer a thorough comprehension of the characteristics and chemical makeup of Calotropis gigantea latex, as well as its potential therapeutic effects. 36 Acta Biologica Slovenica, 2025, 68 (1) Material and Methods Collection of samples Wildly growing Calotropis gigantea (Supplementary Fig. 1) plant samples were collected from roadside locations close to the study area (31.30 latitudes North and 75.60 longitude East). The plant was verified by the botany department, and BT103 was noted on the voucher. This city experiences long, hot summers and chilly winters due to its humid subtropical climate. The study was carried out between January and May of 2024. Preparation of extracts The latex (about 30 mL) was collected from the Calotropis gigantea plant by breaking the stems of the plant with hands and then squeezing the broken stems to collect the latex from them into the Eppendorf tubes and stored at 4 °C for further use. GC-FID analysis of latex Chemical profiling was done using a gas chromatography flame ionization detector (GC-FID) system (Chemtron 2045) for the study of bioactive chemicals (Sharma and Kaur, 2022a). To identify and measure the bioactive chemicals contained in Calotropis gigantea latex, it was mixed with double volumes of isopropyl alcohol/water (8:2 v/v). Shaking the mixture by hand, vortexing it, and letting it lie overnight at room temperature were the next steps before centrifuging it for 15 minutes at 4 °C at 10,000 rpm. After that, the supernatant was collected and subjected to GC-FID examination. A 0.2 µl injection volume of extract was used. A 2-meter-long stainless-steel column fitted with a 10% OV-17 load on an 80–100% mesh Chromosorb W (HP) filter was provided by GC. The carrier gas was nitro- gen gas at a rate of 30 mL per minute. The temperature of the injector and detector was maintained at 200°C and 250°C, respectively. A 0.2μl sample volume was injected. The ramping temperature settings for the oven were 110°C initially, then 200°C at a pace of 2°C per minute. Bioactive compounds were identified by comparing the relative retention duration with published data or standards (Adam, 2007). Ligands Preparation For in-silico docking studies, the predominant phyto- chemicals in Calotropis gigantea latex (Phytol, 4-Methyl-2- phenylindole and α-Tocospiro A) were chosen were ligands for seven bacterial Penicillin-binding protein (PBP1a). The following link was used to retrieve SMILES of ligands: https://pubchem.ncbi.nlm.nig.gov/. The 3-D structures of ligands were generated using UCSF-chimera after SMILES were obtained from the NCBI-PubChem database. Protein preparation Targets bacterial penicillin-binding protein (PBP1a) was the protein involved in the bacterial life cycle and was hence selected as a key target for docking. The RCSB database (https://www.rcsb.org/) was used to retrieve the target mac- romolecule (PDB IDs: PBP1a:3UDF). The PBP1a structure lacks water molecules and natural inhibitors before docking research. The dock-prep function in the Chimera program (chimaera tool 1.14) was used to lower energy. The optimi- zation process of dock preparation takes atomic structure, bond length, and charge anomalies into consideration. Molecular Docking A molecular docking study was performed on the selected phytocompounds using the CB-Dock2 application (https:// cadd.labshare.cn/cb-dock2/php/index.php). The protein receptor (PBP1a) and ligand molecules were then sent to the CB-Dock2 service in pdb file format. A .pdb file with the ideal docked structure was generated and downloaded. In-vitro Anti-bacterial Activity The antimicrobial activity of calotropis latex was assessed using the agar disc diffusion method against four test organisms: G+ Staphylococcus aureus (MTCC 3160), Bacil- lus subtilis (MTCC 121), and G- Escherichia coli (MTCC 40), Pseudomonas aeruginosa (MTCC 424). The microorganisms originate from IMTECH, the Institute of Microbial Technol- ogy in Chandigarh. Calotropis latex (volume: 5-150µl) was impregnated onto sterilized paper discs (5 mm diameter). New inoculums were created using 12-hour-old cultures for each microbial strain. After swabbing the LB-Agar plates with bacterial suspension, they were allowed to dry for thirty minutes. Next, the calotropis latex-impregnated paper discs 37 Acta Biologica Slovenica, 2025, 68 (1) were positioned in the middle of the petri plates. The plates underwent incubation for 24 hours at 37°C after being kept at room temperature for a period of 20 minutes to enable the calotropis latex out of the disc to dissociate into the liquid. Streptomycin (10µg/disc) served as the positive control. After incubation, a zone of inhibition encircling the disc may be observed using a transilluminator; this radius represents the antibacterial activity of calotropis latex. Statistical Analysis The average ± standard deviation is displayed for each study; all investigations were carried out in triplicate using at least three different trials unless otherwise stated. ANOVA (analysis of variance) was utilized with GraphPad Prism v. 6.0 (GraphPad Software, Inc., San Diego, CA, USA) to identify significant differences. Tukey's multiple com- parisons test was employed to evaluate mean differences between groups. Variations were considered significant when p < 0.05. Results and Discussion Gas chromatography analysis Phytochemical profiling is the qualitative test based on colour formations and/or precipitates of herbal formulations as indicators of the presence of secondary metabolites in plant material (Bouzidi et al., 2023). Many contemporary analytical techniques have been developed, and they are crucial in determining the active ingredients, medicinal properties, pharmacological properties, and other details of a substance's application in the fields of biochemical, pharmaceutical, and clinical research. GC-FID phyto- chemical analysis is one of the contemporary methods for identifying and isolating phytoconstituents. One modern technique for identifying and isolating phytoconstituents in plants is GC-FID (Bouzidi et al., 2023; Nkwocha et al., 2023; Onyeike et al., 2023). The typical chromatogram depicting phytochemical compounds present in calotropis latex received by GC-FID is evident in Figure 1. Additionally provided are the noted peaks and the duration of their retention. GC-FID analysis of latex extracted from Calotropis gigantea led to the identification of 13 compounds. Oxygenated diterpenes, i.e. Phytol, with other minor components, were found to be the most dominant phytocompounds at 64%. Acyclic hydrogenated diterpene alcohol, or phytol, is the main component and precursor to synthetic forms of vitamins K and E. It is also directed as phytosol or floral alcohol (Gutbrod et al.,2021). Phytol is used in the production of fragrances. According to Islam et al. (2018), it is also used commercially in shampoos, toilet soaps, detergents, and cosmetics. Additionally, some cannabis distillates use it as a flavouring or dilution. The microscopic peaks could be caused by the bioactive compounds present in trace amounts. The latex composition turned out to be different from previous investigations. Prior research has likewise confirmed the existence of both main and minor elements, albeit with different compositions. For example, in our investigation, phytol accounted for 24% of the total component; in contrast, Al-Rowaily et al. (2020) found that the phytol content was 8.73%. The current findings were entirely consistent with the C. procera EO chemical profile that was published and obtained from the Egyptian delta, where oxygenated diterpene phytol was found in amounts of 38% (Wahba and Khalid, 2018). These findings were consistent with those published for other Egyptian C. pro- cera organs, where the primary chemicals were identified as diterpenes, specifically E-phytol. However, the profile of Indian C. gigantea (Singh and Javed, 2013) showed no diterpenes at all, which was at odds with the current results. Overall, the variance in the metabolites' production, including that of latex, may be the cause of the consider- able disparity in chemical components. Environmental and climatic factors such as temperature, altitude, seasonality, plant age and water availability and soil conditions such as weather, extraction technique, soil moisture, harvest season, the distance between plants, harvest time, drying method, storage conditions, conditions, and genotype, all have a direct impact on the biosynthesis of latex compo- nents (Borges et al., 2018, Amin et al., 2019). Molecular Docking studies In-silico study was conducted by targeting PBP 1(Penicil- lin-binding protein), which is essential for the final steps of peptidoglycan (PG) bacterial cell wall biosynthesis (Galinier et al., 2023). The primary structural element of the bacterial cell wall, PG is necessary to preserve the form, integrity, and survival of the cell. Owing to transpeptidase activity, PBP1 is involved in connecting the peptide side chains of developing glycan strands in cell wall synthesis, thus 38 Acta Biologica Slovenica, 2025, 68 (1) S. No Ret. Time (RT) Bioactive Compound Conc. (%) 1 1.082 Phytol 64.4103 2 3.332 Butanoic acid, 2[(phenyl methoxy)imino] trimethylsilyl ester 4.4500 3 4.082 4-Methyl-2- phenylindole 16.2854 4 18.248 Cyclohexanol,3-methyl 0.0594 5 19.415 Ergost-5-en-3-ol 0.0841 6 21.748 β-Cyclocitral 0.1554 7 30.582 Heptadecyl acetate 0.1362 8 36.415 α-amryin 0.1346 9 38.498 Lup-20(29)-en-3-ol acetate 0.1547 10 45.915 Guanidine nitrate 0.4865 11 52.498 α-Tocospiro A(alpha-tocopheroids) 10.9942 12 57.165 Ursa-12-en-3β-yl pentanoate 2.3091 13 58.332 Astilbin 0.3400 Figure 1. Chemical profile and phytocompounds identified calotropis latex. Green font indicates major compounds used for docking analysis. Slika 1. Kemični profil in identificirane fitokomponente lateksa kalotropisa. Črke v zeleni barvi označujejo glavne spojine, uporabljene za analizo prileganja. being selected as the ideal target for docking studies. The bioactive compounds with the most potential for inhibition are those with the lowest vina score with their targets. According to Agu et al., 2023, molecular docking is an efficient computational method applied here to anticipate and determine the potential binding mode that emerges between a chemical and a specific target protein or recep- tor. Penicillin-binding proteins (PBP1) were used in this study's molecular docking assays, with the most prevalent phytocompounds found in calotropis latex. The docking score of every bioactive molecule against the PBP1 and other pertinent data are displayed in Table 1. 39 Acta Biologica Slovenica, 2025, 68 (1) Ligand Vina score Cavity volume (Å3) Center (x, y, z) Docking size (x, y, z) Contact residues Phytol -5.2 2357 93, 48, -1 27, 27, 27 Chain A: LEU64 ALA65 GLU67 ASP68 SER69 SER70 PHE71 ASN134 TRP205 ARG209 GLN212 LEU213 Chain B: GLN506 MET509 ASP510 PHE511 GLY512 LEU513 GLN514 GLU515 ASP516 LEU532 ILE534 GLN535 THR538 ARG549 VAL550 GLN551 PRO552 4-Methyl-2- phenylindole -7.4 2357 93, 48, -1 29, 20, 32 Chain A: ALA66 GLU67 ASP68 SER69 SER70 PHE71 ASN134 LYS185 ARG202 TRP205 ARG209 GLN212 LEU213 Chain B: LYS255 LEU426 GLN427 TRP429 GLN506 MET509 ASP510 GLY512 LEU513 GLN514 GLU515 ASP516 LEU532 ILE534 GLN535 THR538 ARG549 GLN551 PRO552 α-Tocospiro A -7.9 2357 93, 48, -1 29, 29, 29 Chain A: ALA66 GLU67 ASP68 SER69 SER70 PHE71 ASN134 LEU135 LYS185 ALA186 TYR190 ARG202 TRP205 ARG209 GLN212 LEU213 GLY214 TYR215 Chain B: GLU247 ARG250 SER251 GLU252 VAL254 LYS255 GLU259 LYS421 PHE422 ASN423 LEU426 GLN427 TRP429 MET509 ASP510 GLY512 LEU513 GLN514 GLU515 LEU532 ILE534 GLN535 THR538 ARG549 GLN551 PRO552 GLU572 GLN631 ILE633 Phytol -5.2 2357 93, 48, -1 27, 27, 27 Chain A: LEU64 ALA65 GLU67 ASP68 SER69 SER70 PHE71 ASN134 TRP205 ARG209 GLN212 LEU213 Chain B: GLN506 MET509 ASP510 PHE511 GLY512 LEU513 GLN514 GLU515 ASP516 LEU532 ILE534 GLN535 THR538 ARG549 VAL550 GLN551 PRO552 4-Methyl-2- phenylindole -7.4 2357 93, 48, -1 29, 20, 32 Chain A: ALA66 GLU67 ASP68 SER69 SER70 PHE71 ASN134 LYS185 ARG202 TRP205 ARG209 GLN212 LEU213 Chain B: LYS255 LEU426 GLN427 TRP429 GLN506 MET509 ASP510 GLY512 LEU513 GLN514 GLU515 ASP516 LEU532 ILE534 GLN535 THR538 ARG549 GLN551 PRO552 α-Tocospiro A -7.9 2357 93, 48, -1 29, 29, 29 Chain A: ALA66 GLU67 ASP68 SER69 SER70 PHE71 ASN134 LEU135 LYS185 ALA186 TYR190 ARG202 TRP205 ARG209 GLN212 LEU213 GLY214 TYR215 Chain B: GLU247 ARG250 SER251 GLU252 VAL254 LYS255 GLU259 LYS421 PHE422 ASN423 LEU426 GLN427 TRP429 MET509 ASP510 GLY512 LEU513 GLN514 GLU515 LEU532 ILE534 GLN535 THR538 ARG549 GLN551 PRO552 GLU572 GLN631 ILE633 Table 1. Molecular docking of α-amylase with the bioactive component of latex derived as ligand. Tabela 1. Molekularno prileganje α-amilaze z bioaktivno komponento lateksa kot ligandom. *Green font indicates common residues among ligands involved in interactions. Figure 2 shows 3D models of the PBP1 complex with affini- ties ligands and their interactions. With vina scores ranging from -5.2 to -7.9, the three common phytochemicals found in the latex were demonstrated to have binding potentials. α-Tocospiro-A exhibited the most efficient binding of all the bioactive components, with a docking score of -7.9, followed by 4-Methyl-2- phenylindole (vina core -7.4) and phytol (vina score -5.2). The ligand's affinity for the receptor is determined by its ability to create hydrogen bonds or hydrophobic interactions with the receptor protein's active site residues during docking (Sharma and Kaur, 2022b). It was observed that almost all phytochemicals interacted with the PBP1a receptor via Van der Waal, alkyl, and Pi-alkyl interactions (Table 1). Notable all phytocompounds involved some common residues located at A and B chains to interact with the PBP1a receptor, as shown in Table 1. Antibacterial agents have a wide range of targets at which they can prevent the formation of cell walls. For many years, these systems have been regarded as significant targets for antimicrobial agents (Bruning et al., 2011). It has been dis- covered that natural substances prevent PBP from forming cell walls. The transpeptidase action of PBP is attributed to the C-terminal module (Contreras-Martel et al. 2017). Studies have demonstrated that therapies that mimic the structure 40 Acta Biologica Slovenica, 2025, 68 (1) of the D-peptide moiety, D-body, such as β-lactam antibi- otics, can obstruct the processes of transpeptidation or carboxypeptidation, which depletes peptidoglycan and may even result in cell death (Da-Costa et al. in 2018). This results in decreased cell wall integrity and the inability to transfer peptidoglycan (Moon et al., 2018). The results of Sripathi and Ravi (2017) reported that molecular docking analyses to determine the antibacterial efficiency of the most active components of Plectranthus hadiensis essential oil against PBPs are consistent with our findings. Additionally, molecu- lar docking investigations of Pogostemon cablin essential oil components against PBPs were published by Yang et al. (2013). The study's findings suggest that plant-based metab- olites might be the primary source of antibacterial chemi- cals. Based on the aforementioned observations, it can be inferred that phytocompounds exhibited a strong affinity for PBP1a upon ligand binding. Consequently, it was postulated that PBP1a alteration results in a modification of the bacterial cell wall and enzyme conformation. By stopping bacterial viability, all of these occurrences lessen the bacteria's capacity to invade the host cell. Previous research has also shown through in-silico results that multi-pharmacological drugs have antibacterial properties that can regulate the proliferation of microbial strains (Lima et al., 2019). Figure 2. 2D models and 3D interactions of phytocompounds with PBP1a receptor. Slika 2. 2D-modeli in 3D-interakcije fitokomponente z receptorjem PBP1a. 41 Acta Biologica Slovenica, 2025, 68 (1) Antibacterial potential of latex The wet lab experiment study was designed to assess the anti-bacterial potential of calotropis latex against four bacterial strains to validate the in-silico findings. According to the current analysis, calotropis latex exhibited potent antibacterial activity comparable to synthetic antibacterial drugs. Table 2 and Supplementary Figure 1-5 present the findings. At 150µl, calotropis latex showed very strong antibac- terial activity against P.aeruginosa (MTCC 424 G-) and Staphylococcus aureus (MTCC 3160 G+) with inhibition zone (IZ) of 2.2cm and 1.6cm, respectively. E.coli (MTCC 40 G-) and B.subtilis (MTCC 121 G+) exhibited moderate inhibi- tory activity with IZ values of 1.1 cm and 1.2 cm, respectively. The potent antibacterial properties of calotropis latex could be attributed to the presence of major and minor bioac- tive components in toto that suppressed partners such as microbial adhesions, cell wall envelop proteins, and non-specific interactions with carbohydrates, or affected the inhibition of hydrolytic enzymes (proteases) (Saddiq et al. (2022). Calotropis latex may have multiple targets or a single target, contributing to its varying antibacterial activity against different bacterial strains. Additionally, prior research has shown that the synergistic effects of major and minor components, rather than the high concentration of a single chemical compound, are often responsible for anti-microbial activity (Vaou et al., 2022). Previous studies have shown that anti-microbial activity can occasionally be attributed to the combined effects of major and minor chemical components as opposed to a high concentration of a single main chemical molecule (Vaou et al., 2022). Microbial culture Volume (µl) Inhibition zone (cm) Escherichia coli MTCC 40 NC - PC 2±0.02a 5 NI 20 0.7±0.03c 50 0.9±0.03c 150 1.1±0.04c Bacillus subtilis MTCC121 NC - PC 1.5±0.01d 5 NI 20 0.6±0.03c 50 0.9±0.02c 150 1.2±0.02c Pseudomonas aeruginosa MTCC424 NC - PC 1.6±0.02d 5 NI 20 1±0.02c 50 1.3±0.04d 150 2.2±0.02e Staphylococcus aureus MTCC3160 NC - PC 1.7±0.01d 5 NI 20 0.5±0.02c 50 1.0±0.03c 150 1.6±0.04d Table 2. Anti-bacterial activity of calotropis latex of different microbial cultures. Tabela 2. Protibakterijsko delovanje lateksa kalotropisa na različne mikrobne kulture. Note: PC: Positive Control, NC: negative control (Streptomycin 10 µg); NI: no inhibition. Results are expressed as Mean ± SD of three determinations (n = 3). Mean values with different letters (a-d) across the columns are considered significantly different at p < 0.05. 42 Acta Biologica Slovenica, 2025, 68 (1) Nejhad et al.'s study from 2023 also mentioned the antibacterial activity of an aqueous extract of Calotropis procera leaves against E. coli, but IZ was only 14.1 mm. One significant finding from our study's CEO was that G-bacteria were highly toxic to antibiotics. This is notable because earlier studies have suggested that G-bacteria's thicker cell walls allow them to express resistance to drugs, and antibiotics in particular, making them more resistant than G+ bacteria (Saxena et al., 2023). The chemical compo- nents found in the plant combination are often responsible for the antibacterial action of a plant extract. Rather than coming from the presence of a single major active element, a medicinal plant's optimum effectiveness may come from the combination of multiple diverse ingredients (Nejhad et al., 2023). The primary cause of the variations in antibacte- rial activity is the plant's varied concentration of secondary metabolites, which may be impacted by elements including species and extraction techniques. Compared to G+ bac- teria, which have a single cell wall structure, G- bacteria enclose a multilayered cell wall structure, increasing their resistance to antimicrobial agents (Nooshkam et al., 2019; Shahidi et al., 2020). In recent times, there has been a growing significance placed on the antibacterial properties of plant flavonoids against various microbial species. Plant extracts typically have antimicrobial properties because they contain phenolic compounds with free hydroxyl groups, like flavonoids. These substances can disrupt bacterial membrane permeability, change cellular peptido- glycan, bind to the active site of enzymes, form hydrogen bonds with enzymes, and disrupt soluble and extracellular proteins, glutamate, and phosphate of bacteria, among other mechanisms (Shahidi et al., 2020). Conclusion Antibiotic resistance to both G+ and G- bacteria is now present in human populations and might endanger health around the globe. PBP's are currently the primary focus of infection. The goal of this work was to examine the bio- active components of calotropis latex that may be utilized to block bacterial infection pathways. Effective docking of all major phytocompounds with PBP1 and antibacterial activity has been demonstrated by docking and in vitro investigations. Therefore, we suggest that calotropis latex, which is included in herbal medications, might be a viable treatment choice and function as a bacterial PBP inhibitor. To make space for these compounds in drug development, more research utilizing in vitro and in vivo models should be carried out to validate these compounds. Funding Funding: Dept of Science and Technology, Govt. of India, DST/SEED/SCSP/STI/2019/253 Author's contributions Conceptualization, ADS; methodology, AC.; software, AC; validation, ADS.; formal analysis, ADS and AC; investiga- tion, ADS; writing—original draft preparation, ADS; writ- ing—review and editing, ADS; visualization, AC; supervi- sion, ADS; project administration, ADS; funding acquisition, ADS. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare no conflict of interest. 43 Acta Biologica Slovenica, 2025, 68 (1) References Agu, P.C., Afiukwa, C.A., Orji, O.U., Ezeh, E.M., Ofoke, I.H., Ogbu, C.O., Ugwuja, E.I., Aja, P.M., 2023. Molecular docking as a tool for the discovery of molecular targets of nutraceuticals in diseases management. 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Alsaadi 2, Zainab Amer Hatem 2, Saade Abdalkareem Jasim 3* Abstract Escherichia coli is the most common type of Enterobacteriaceae family that causes urinary tract infections. This bacterium causes diseases due to its many virulence factors. During the study, (150) urine samples were collected for women with symptoms of UTIs aged between (20-50) years from 1/9/2021 to 1/4/2022. Forty- five isolates of E. coli were diagnosed using a biochemical test, a VITEK2 system, and molecular detection using traditional PCR. A molecular study was conducted on 25 of these isolates using conventional PCR and qRT-PCR to investigate the presence of genes included (16S rRNA, uidA, fimA and kpsMTII). The traditional PCR results showed the presence of both 16S rRNA and uidA gene in all 25 isolates of E. coli. In contrast, the results showed that 23 (92%) and 18 (72%) isolates possessed fimA and kpsMTII genes, respectively. Following the extraction of RNA from twenty-five pathogenic isolates of E. coli and ten non-pathogenic isolates, gene expression of fimA and kpsMTII was quantified using quantitative real- time reverse transcription PCR (qRT-PCR). The observed fold change indicates a potential overexpression of the KSPMII and FimA genes in uropathogenic E. coli. The expression of the kpsMTII gene showed the highest level of folding with an average of 22.89 in the case of uropathogenic E.coli isolates compared with non-pathogenic isolates (control) with an average of 4.11. While the expression of the fimA gene with the highest level of fold change with an average of 90.52 in the case of uropathogenic E.coli isolates compared with non-pathogenic isolates (control) with an average of 0.94, Significant differences were observed among the isolates, as indicated by P values less than 0.05. This finding demonstrated that qRT-PCR detects genes that were expressed in comparison with traditional PCR. Also, qRT-PCR is a more sensitive and accurate assay than conventional PCR for determining the quantitative prevalence of E. coli in urine samples. Keywords UPEC (uropathogenic E. coli); qRT-PCR; fimA, kpsMTII 1 College of Education for Pure Sciences, University of Diyala, Diyala Gov, Iraq 2 College of Sciences, University of Diyala, Diyala Gov, Iraq 3 Medical Laboratory Techniques Department, College of Health and Medical Technology, University of Al-maarif, Anbar, Iraq * Corresponding author: E-mail address: saade.a.j@uoa.edu.iq Citation: Mahdi, Z. H., Alsaadi, L. A. S., Hatem, Z. A., Abdalkareem, S., (2024). Qualitative and Quantitative Detection of Virulence-Associated Genes in Escherichia Coli Isolates from Women with Urinary Tract Infections. Acta Biologica Slovenica 68 (1) Received: 21.08.2024 / Accepted: 26.11.2024 / Published: 04.12.2024 https://doi.org/10.14720/abs.68.01.19574 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license 46 Acta Biologica Slovenica, 2025, 68 (1) Uporaba klasičnega PCR in RT-PCR za odkrivanje nekaterih genov faktorjev virulence bakterij Escherichia coli, izoliranih iz pacientk z uroinfektom Izvleček Escherichia coli je najpogostejša vrsta iz družine Enterobacteriaceae, ki povzroča okužbo sečil. Ta bakterija lahko povzroči bolezni zaradi številnih dejavnikov virulence. Med študijo je bilo zbranih 150 vzorcev urina pri ženskah s simptomi infekcije sečil v starosti 20-50 let. Vzorci so bili nabrani v obdobju od 1.9.2021 do 1.4.2022. Diagnostično smo uporabili 45 izolatov E. coli z biokemičnim testom, sistemom VITEK2 in molekularno detekcijo s klasičnim PCR. Molekularna študija je bila izvedena na 25 od teh izolatov z uporabo klasičnega PCR in qRT-PCR za gene 16S rRNA, uidA, fimA in kpsMTII. Rezultati PCR so pokazali prisotnost 16S rRNA in gena uidA v vseh 25 izolatih E. coli, medtem ko so rezultati pokazali, da je imelo 23 (92 %), izolatov gen fimA in 18 (72 %) gen KSPMII. Po ekstrakciji RNA iz petindvajsetih patogenih izolatov E. coli in desetih nepatogenih izolatov smo ekspresijo genov kvantificirali s kvantitativno PCR v realnem času (qRT-PCR). Opazili smo potencialno čezmerno izražanje genov KSPMII in FimA v uropatogeni E. coli. Ekspresija gena KSPMII je pokazala najvišjo stopnjo ekspresije s povprečjem 22,89 v primeru uropatogenih izolatov E.coli v primerjavi z nepatogenimi izolati (kontrola) s povprečjem 4,113. Medtem ko smo najvišjo ekspresijo gena fimA s povprečjem 90,52 izmerili v primeru uropatogenih izolatov E.coli. Povprečje za nepatogene izolate (kontrola) je bilo 0,946. Naša raziskava je pokazala, da je qRT-PCR bolj občutljiv in natančen test kot klasični PCR za določanje kvantitativne prevalence E. coli v vzorcih urina. Ključne besede UPEC (uropatogena E. coli); qRT-PCR; fimA, kpsMTII Introduction The term urinary tract infections (UTIs) refer to the presence of bacteria which are not within the normal microbiota of the vagina, urethra and skin in the urinary tract environment with a significant number (≥105) cells/ml in the middle urine sample (Nicolle et al., 2019), these infections are a health problem in both the community and hospitals (Ahmed et al., 2019). In females, they are more common than males due to a variety of variables that occur in the female body, espe- cially the urinary tract, such as shortness of the urethra and entry facilitated by pathogens through sexual contact, and the proximity of the anus to the vagina (Ahmed and Yousry, 2021), This area surrounding the rectum is hot and humid, which is teeming with living organisms, where bacteria can easily reach the bladder in females (Tille, 2014). The main culprit of UTIs is the Enterobacteriaceae family, the most common of which is Escherichia coli, which is responsible for about (90-80%) of all these infections worldwide (Zal- manovici et al., 2010; Dhahi, 2020). E. coli possesses sev- eral virulence factors that enable it to colonize and induce infection in the urinary tract, including the ability to adhere to urethral epithelial cells encoded by (sfa, fimA and pap). Alpha-hemolysin is a protein extracellular cytolytic toxin encoded by hlyA gene, whereas the kpsMTII gene encodes for capsule synthesis, the ompT gene encodes for protease in outer membranes, and has iron acquisition systems encoded by (fyuA, ironN, iucD and iutA). Virulence-associ- ated genes (VAGs) are either carried on the chromosome or the plasmid (Dadi et al., 2020). Rapid and accurate diagnosis is a step towards treatment, and is done by using molecular techniques, including polymerase chain reaction (PCR) and quantitative real-time reverse transcription (qRT-PCR) tech- niques. The technique to screen bacteria, including Esche- richia coli, relies on determine the possession of the gene encoding virulence factors and enhancing epidemiological information when investigating this bacterium (Maniam et al., 2022). This study aimed to use molecular detection by traditional PCR and qRT-PCR for analysing the presence of diagnostic genes included (16S rRNA and uidA) and some virulence factors genes (fimA, kpsMTII) of E. coli isolated from UTIs of women in Diyala/Iraq. 47 Acta Biologica Slovenica, 2025, 68 (1) Materials and Methods Bacterial Isolation One hundred and fifty samples were taken from women with symptoms of UTIs their ages between (20-50) years from 1/9/2021 to 1/4/2022, Samples were collected from two hospitals in Diyala, Iraq, including Baquba General Hospital and Al-Batol Hospital for maternity and children. Forty-five isolates of E. coli were obtained. After cultured sample on multiple media, including Blood agar, MacConkey agar, and Chrom agar Orientation agar. The initial identification depended on conventional methods (biochemical test) and the VITEK2 system (bioMerieux, France). We used traditional PCR to detect the 16S rRNA and uidA genes for identification confirmation. Detection of studied genes by traditional PCR The instructions of the producing companies were applied for both the DNA extraction for isolates prepared from (Geneaid Thailand) and the preparation of the studied gene primers prepared from Macrogen (USA) for detection genes, including (16S rRNA, uidA, fimA and kpsMTII). The primers for the studied genes were clarified in Table 1. The mixture for the traditional PCR reaction for all genes consists of 1 μl forward and reverse primers for each gene at a concentration of 10 pmol/μl, two μl DNA, and 8.5 μl nuclease-free water to the microtubules containing the components (i-Taq) The final volume of the PCR reaction was 25 μl. The Thermal cycle was programmed individually for each gene, as shown in Table 2. Agarose gel was used for gene electrophoresis. After the migration expired, the stained bundles were visualized by a gel imaging system. Extracted RNA from bacterial isolates was done accord- ing to the manufacturer's instructions using an extraction kit(Quick-RNA™, USA). Unique spin-column technology is used to produce high-quality total RNA (including small/ microRNAs) prepared for RT/qPCR. EasyScript® First- Strand cDNA Synthesis SuperMix. To conduct the quan- titative (qRT-PCR) by using Syber Green chemistry, the LunaScript qRT Master Mix Kit (5X) from BioLabs (England) was utilized. The reaction mixture was prepared with a total volume of 20 µl, consisting of 10 µl of the master mix, 0.5 µl each of the forward and reverse primers, 5 µl of cDNA from each sample, and 4 µl of nuclease-free water. The reaction was mixed thoroughly and centrifuged briefly to collect solutions at the bottom of the PCR tubes. Then, after the preparation, the tubes were placed into the thermal cycler programmed in Table 3. The qPCR protocol was initiated with polymerase activation at 95°C for 1 minute. This was followed by 45 amplification cycles, each consisting of a denaturation step at 95°C for 15 seconds and an annealing step at 60°C for 20 seconds, during which channel scan- ning was performed. Melting curve analysis was conducted based on the separation characteristics of double-stranded cDNA during cycles with progressively increasing dena- turation temperatures. The Ct values of the target genes (kpsMTII and fimA) were normalized to the 16S RNA refer- ence gene, and The expression levels of the target genes were quantified using the relative quantification approach, utilizing the comparative Ct method combined with fold change analysis, precisely calculated as 2^-ΔΔCt. Primers Name Primer Sequence Product size (bp) Reference 16S rRNA F: AGA GTT GAT CCT GGC TCA 1500 (Maleki et al., 2017) R: GGT TAC CTT GTT ACG ACTT uidA F: CAT TAC GGC AAA GTG TGG GTC AAT 658 (Adamus-Bialek et al., 2009) R: CCA TCA GCA CGT TAT CGA ATC CTT fimA F: GTT GTT CTG TCG GCT CTG TC 447 (Hasan et al., 2021) R: ATG GTG TTG GTT CCG TTA TTC kpsMTII F: GCG CAT TTG CTG ATA CTG TTG 292 (Johnson and Stell, 2000) R: CAT CCA GAC GAT AAG CAT GAG CA Table 1. The primers of the studied genes. Tabela 1. Primeri preučevanih genov. 48 Acta Biologica Slovenica, 2025, 68 (1) Results The results of this study obtained 45(30%) E. coli isolates from women suffering from UTIs using a conventional VITEK2 system and molecular methods for identification After obtaining ethical approval to take urine samples from patients. A molecular study was conducted on 25 of these isolates using traditional PCR and qRT-PCR to investigate the presence of genes included (16S rRNA, uidA, fimA and kpsMTII). The conventional PCR results showed the presence of (16S rRNA and uidA) which diagnostic gene in all 25 isolates of E. coli. In contrast, 23 (92%) and 18 (72%) isolates possessed fimA and kpsMTII genes, respectively. Molecular detection results for all genes using traditional PCR are presented in Table 4. A significant statistical differ- ence (p < 0.05) between isolates carrying each gene and isolates was observed for fimA and kpsMTII. Real-time qRT-PCR quantification of kpsMTII and fimA gene expression The quantitative qRT-PCR reaction experiment used (25) pathogenic and (10) non-pathogenic isolates. Melting curve analysis was carried out based on the separation properties of double-stranded cDNA during cycles with progressively increasing denaturation temperatures (Tm). The assay was conducted in triplicate for each sample, and the average of these three measurements was used to quantify the gene expression for that sample. Ct value of target genes (kpsMTII and fimA) had been standardized to 16sRNA reference genes, and the relative quantitative method was used to determine the target. To represent gene expression using the comparative Ct formula and Fold change analysis (2^-ΔΔCT), you can create a table with the following figure (5). Expression of fimA and kpsMTII in the pathogenic E. coli was significantly higher than in non-pathogenic strains (Fig. 1). The expression of the kpsMTII gene exhibited the high- est level of upregulation, with a mean value of 22.8943, in uropathogenic E. coli isolates compared to non-pathogenic isolates (control), with a mean value of 4.113. Statistical anal- ysis revealed significant differences between the groups, with P-values < 0.05. Figure 1 shows the expression of the fimA gene with the highest level of fold change with an average of 90.52 in the case of uropathogenic E. coli isolates compared with non-pathogenic isolates (control) with an average of 0.946. Significant differences between the isolates were observed, with P values < 0.05. Genes Denaturation Annealing Extension* No. Cycles 16S rRNA 94ºC / 30 sec 55 ºC / 30 sec 72 ºC/30 sec 35 uidA 92ºC/60 sec 58 ºC / 60 sec 72 ºC/30 sec 35 fimA 94ºC/45 sec 55 ºC / 45 sec 72 ºC/54 sec 30 KPSMTII 94ºC/30 sec 60°C/ 60 sec 72ºC/60 sec 30 qPCR Steps Temp. Time Cycle(s) Initial activation 95ºC 10min 1 Denaturation 95ºC 15 sec 45 Annealing 60ºC 20 sec Extension 90ºC 15 sec Table 2. PCR program for studied genes. Tabela 2. Program PCR za preučevane gene. Table 3. qRT-PCR program. Tabela 3. Program qRT-PCR Initial Denaturation 94ºC\ 5 min and final extension 72°C\ 10 min for all genes. * 49 Acta Biologica Slovenica, 2025, 68 (1) 16SrRNA uidA fimA kpsMTII No. isolates 25 25 25 25 Carry gene (+) 25(100%) 25(100%) 23(92%) 18(72%) Not carry gene 0 (0%) 0 (0%) 2 (8%) 7(28%) P value 1.00 1.00 P<0.001*** P<0.001*** Groups N ΔCT (Mean±SE) ΔΔCT (Mean±SE) Fold Change (2^- ΔΔCT) Mean±SE Control kpsMTII 10 7.01±0.76 5.00± 0.31 4.11±0.45 Control fimA 10 18.21±1.46 11.36±1.04 0.94±0.11 kpsMTII 25 6.67±0.94 -0.42±0.12* 22.89±1.62** fimA 25 14.23±1.04 -4.44±0.34 90.52±3.46** Table 4. Gene prevalence used traditional PCR. Tabela 4. Razširjenost genov, uporabljenih pri tradicionalni PCR. Table 5. The gene expression of kpsMTII AND fimA in Pathogenic and non-pathogenic E.coli. Tabela 5. Izražanje genov kpsMTII IN fimA v patogenih in nepatogenih E.coli. Figure 1. Folding change of kpsMTII and fimA gene expression. Slika 1. Spremembe prepogibanja kpsMTII in izražanje gena fimA. 50 Acta Biologica Slovenica, 2025, 68 (1) Discussion Urinary tract infections are the most common bacterial infec- tion, affecting all age groups, especially women, E. coli is the most common cause, followed by Klebsiella (Abalkhail et al., 2022). The results obtained in this study are consistent with the results of other studies, including Thanoun (2022) from Mosul, Ahmed et al. (2019) from Saudi Arabia, and Mathai et al. (2001) from America, if the percentage of infection with this bacteria was 33.1%, 27%, 46.9%, respectively. Molecular results for 16SrRNA and uidA genes using tra- ditional PCR explained that all isolates possess them, which also agrees with the researcher Salih (2015). Therefore, it can be considered an effective diagnostic method for identifying the presence of E. coli in patients with urinary tract infections, confirmed through isolation and subsequent analysis. This was similared withe Forbes et al. (2007) and Brooks et al. (2010) that the final diagnosis of E. coli was made based on molecular methods, especially the 16SrRNA gene. The uidA gene is a frequently used enzyme marker for detecting these bacteria in newly developed detection media (Brons et al., 2020) as this gene encodes an enzyme specific to E. coli, the enzyme beta-D-glucuronidase (Cleuziat and Robert-Bau- douy, 1990). This gene is used to diagnose UPEC, and the results of this study were confirmed to those of Brons et al. (2020), in which all isolates possessed uidA gene. All twenty-five pathogenic isolates expressed target genes (kpsMTII and fimA) by using qRT-PCR in different levels of fold change, While by using traditional PCR, the results showed that 23(92%), 18(72%) of isolates pos- sessed fimA and kpsMTII genes respectively. This result demonstrated that the qRT-PCR technique was Quanti- tative PCR (qPCR) demonstrates superior sensitivity and specificity compared to conventional PCR for assessing the presence of E. coli in UTI specimens. The fimA adhesion virulence gene in these isolates is implicated in enhancing the colonization and persistence of E. coli in the urinary tract. Additionally, the expression of kpsMTII genes, which are crucial for capsule synthesis, was significantly elevated in these isolates. These genes have been associated with increased pathogenicity, a higher probability of antibiotic resistance, and biofilm formation. Implementing qRT-PCR for some genes in clinical practice may facilitate the identi- fication of individuals at elevated risk for (UTIs) with UPEC and contribute to reducing antibiotic usage. The creation of UTI in the host is made more accessible by the UPEC's capacity to acquire the genes that code for virulence, allowing it to persist continue in the urinary system. Consequently, the development and expression of the genes that code for virulence are frequently linked to the severity of clinical UTI symptoms (Stork et al., 2018). Identi- fying UPEC-associated virulence genes in E. coli strains can inform the decision-making process regarding the necessity of antibiotic treatment and predict the likelihood of asymp- tomatic bacteriuria progressing to symptomatic urinary tract infections (UTIs). This is crucial as evidence suggests that bacterial colonization may play a role in preventing symp- tomatic UTIs (Salvador et al., 2012; Maniam et al., 2022). All these factors emphasis the importance need to identify the specific characteristics of the organism, including its virulence traits, before initiating therapy with E. coli strains. qRT-PCR is a highly sensitive and powerful method for analyzing gene expression levels of genes. Comprehending its technical components is necessary to utilise its quantita- tive capabilities fully. Quantitative qRT-PCR necessitates the correction of experimental differences in individual qRT and PCR efficiency for successful results (Pakbin et al., 2023). Conclusions Quantitative Real-Time PCR (qRT-PCR) utilizing SYBR Green melting curve analysis has proven to be a highly sensitive and specific method with a low detection threshold for identifying and quantifying E. coli isolates in urine samples. Author Contributions Conceptualization, Z.A.H; methodology, Z.A.H.; software, L.A.S.A.; validation, Z.H.M.; investigation, S.A.J.; writin – original draft preparation, Z.H.M.; writing – review and editing, L.A.S.A.; visualization, S.A.J.; supervision, Z.H.M. All authors have read and agreed to the published version of the manuscript. Acknowledgement Thanks and gratitude to the University of Diyala for its con- tinuous support to researchers, as well as the University of Almaarif for facilitating the task of completing the research in its laboratories. 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Rev,10:CD007182. 52 1 Laboratory of Botany and Valorization of Plant Diversity, Doctoral School of Sciences, Technologies and Environment, Natural Sciences Training and Research Unit, Nangui Abrogoua University of Abidjan, 02 BP 801 Abidjan 02, Côte d'Ivoire. 2 Department of Agriculture New Technologies, Fisheries Resources and Agro-industry, Training and Research Unit in Agriculture, Polytechnic University of San Pedro, BP V1800 San Pedro, Côte d’Ivoire. 3 Laboratory of Plant Biology and Earth Sciences, Science and Technology Training and Research Unit, Alassane Ouattara University of Bouaké, 01 BP 18 Bouaké 01, Côte d’Ivoire. * Corresponding author: E-mail address: kyabdoulrahimfalk@gmail.com Citation: Ky, A. R. F., Konaté , M. L., Kouamé, N. F., (2024). Impact of water deficit on the anatomical structure of more productive and less productive cashew trees (Anacardium occidentale L.) in Côte d’Ivoire. Acta Biologica Slovenica 68 (1) Received: 13.11.2024 / Accepted: 05.12.2024 / Published: 28.10.2024 https://doi.org/10.14720/abs.68.01.19604 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Impact of water deficit on the anatomical structure of more productive and less productive cashew trees (Anacardium occidentale L.) in Côte d’Ivoire Ky Abdoul Rahim Falk 1*, Konaté Mory Latif 2, Kouamé N’Guessan François 3 Abstract This study aimed to understand the mechanism of resilience of the anatomical tissues of organs of the cashew tree in water deficit in the different climatic zones of Côte d'Ivoire. To achieve this, samples of leaves and roots were taken from 55 cashew trees considered high producers (APHP) and 164 neighbouring less productive trees (non-APHP) during the dry and rainy seasons. The technique of double staining with green-carmino made it possible to obtain anatomical sections of transversely cut samples under an optical microscope. The thicknesses and surface areas of the sections were measured using Image J software. The thickness of the leaf parenchymas of APHP trees did not show any variation according to the season, while the stomatal density increased during the dry season. The thickness of the leaves cortical and medullary parenchymas of non-APHP trees did not show any variation according to the season too, but the non-APHPs’ palisade parenchyma thickness increased while their lacunar parenchyma thickness decreased from 37 ± 1.5 µm to 36 ± 3.6 µm during the dry season. The thickness of leaves on both sides of the epidermis increased while the thickness of xylem bundles decreased during the dry season in both APHP and non-APHP trees. The number of the roots’ xylem vessels of APHP trees increased from 11 to 27 vessels/mm2, while those of non-APHP trees increased from 15 to 41 vessels/mm2 during the rainy season. The surface area of the midrib medullary parenchyma of APHP trees increased from 4300 ± 837 µm2 to 5800 ± 412 µm2 while those of non-APHP trees increased from 3400 ± 809 µm2 to 5200 ± 993 µm2 during the dry season. The thicknesses of the tissues of APHPs remained greater than those of non-APHPs, regardless of the sampling season. Keywords Cashew tree, anatomy, adaptation, water deficit, Côte d’Ivoire 53 Acta Biologica Slovenica, 2025, 68 (1) Vpliv pomanjkanja vode na anatomsko strukturo bolj produktivnih in manj produktivnih dreves indijskega oreščka (Anacardium occidentale L.) v Slonokoščeni obali Izvleček Namen naše raziskave je bil razumeti mehanizem odpornosti anatomskih tkiv organov drevesa indijskega oreščka na pomanjkanje vode v različnih podnebnih območjih Slonokoščene obale. Za dosego tega cilja so bili med suho in deževno sezono odvzeti vzorci listov in korenin pri 55 drevesih indijskega oreščka, ki veljajo za visoko produktivna (APHP), ter pri 164 sosednjih manj produktivnih drevesih. Tehnika dvojnega barvanja z zelenim karminom je omogočila pridobitev anatomskih prerezov prečno rezanih vzorcev, ki smo jih opazovali pod optičnim mikroskopom. Debeline in površine prerezov so bile izmerjene z uporabo programske opreme Image J. Debelina parenhima listov pri drevesih APHP ni kazala sezonskih sprememb, medtem ko se je gostota rež povečala v sušni sezoni. Prav tako se debelina kortikalnega in medularnega parenhima listov dreves brez APHP ni spreminjala glede na sezono, vendar se je debelina njihovega palisadnega parenhima povečala, medtem ko se je debelina lakunarnega parenhima zmanjšala s 37 ± 1,5 μm na 36 ± 3,6 μm v sušni sezoni. Debelina epidermisa na obeh straneh listov se je povečala, medtem ko se je debelina ksilema zmanjšala v sušni sezoni pri drevesih APHP in ne-APHP. Število ksilemskih elementov v koreninah dreves APHP se je povečalo z 11 na 27 žil/mm², pri drevesih brez APHP pa s 15 na 41 žil/mm² v deževni sezoni. Površina medularnega parenhima glavne žile korenin pri drevesih APHP se je povečala s 4300 ± 837 μm² na 5800 ± 412 μm², medtem ko se je pri drevesih brez APHP povečala s 3400 ± 809 μm² na 5200 ± 993 μm² v sušni sezoni. Debeline tkiv pri drevesih APHP so ostale večje kot pri drevesih brez APHP, ne glede na sezono vzorčenja. Ključne besede Indijski orešček, anatomija, prilagoditev, pomanjkanje vode, Slonokoščena obala Introduction The cashew tree (Anacardium occidentale L.) belongs to the clades Angiospermae and Eudicotidae, the order Sap- indales and the Anacardiaceae family (APG 2016). Cashew cultivation constitutes an important new source of income for African farmers, who account for more than 55% of the global cashew nut production. Côte d’Ivoire is the leading African producer and exporter of raw cashew nuts (Henry et al. 2011) and the world’s third biggest producer and exporter of raw cashew nuts after India and Vietnam (FAO 2024). Its production of raw cashew nuts was estimated at 970,000 tons in 2022 (FAO 2024). Despite this performance and constant evolution, Ivorian orchard yields are between 350 to 500 kg/ha (Djaha et al. 2010, FAO 2024) compared to those obtained in India, Vietnam, Brazil and Tanzania, which are between 1,000 and 1,500 kg/ha (Kiwuso et al. 2013). Cashew nut producers encounter enormous diffi- culties due to several factors, such as insufficient technical supervision, the use of low-yielding varieties and unknown sources (Charahabil et al. 2017). In Côte d’Ivoire, the mass selection technique was used to identify Potentially High-Producing APHP Cashew Trees (Konan 2016). These trees were selected on the basis of the following characteristics: good tree architec- ture, precocity and grouped maturity, shape and quality of nuts, nut weight ≥ 7 g and yield ≥ 10 kg of nuts/tree/year. However, some studies have pointed out the existence of a relationship between the anatomy and the production potential of fruit trees. However, abiotic factors are the trigger for stress. Plant responses to these stresses are either plastic and reversible or irreversible (Skirycz and Inze 2010, Cramer et al. 2011). Indeed, the leaf is the most adaptable organ in its response to environmental condi- tions (Marchi et al. 2008). Leaf structures reflect the effects of water stress more clearly than the structures of stems and/or roots, whose effects are also visible. This work aimed to understand the resilience mecha- 54 Acta Biologica Slovenica, 2025, 68 (1) nisms of leaf and root tissues of Potentially High-Producing Cashew Trees (APHP) and their neighbours (non-APHP) in the cashew basin of Côte d'Ivoire according to different seasons. Material and methods Study areas This study was carried out in the cashew basin of Côte d'Ivoire across 15 administrative regions (Figure 1) and two agro-ecological zones, characterized as follows: • Guinean Sector, which is a transition zone between the forest zone in the south and the grassy savannah of the centre, with a dry season from January to February, a major rainy season from March to October and a small rainy season from November to December (FAO 2005, Konaté 2021). The annual rainfall is between 1,031.5 mm and 1,189.36 mm, while the temperature varies between 16 °C and 36 °C, with an average of 27 °C (FAO 2005, Ouattara et al. 2016); • The Sudanian Sector is characterized by the north savannah vegetation, with a dry season from November to February and a long rainy season from March to October (FAO 2005, Konaté 2021). The annual rainfall is between 1,316.19 mm and 1,708.39 mm, and the tem- perature varies between 28 °C and 32 °C (FAO 2005, Ouattara et al. 2016). Figure 1. Map of the sample collection area. Slika 1. Zemljevid območja zbiranja vzorcev. 55 Acta Biologica Slovenica, 2025, 68 (1) Data collection The samples of mature leaves and roots were collected according to the method of Sankharé (2018) from 219 cashew trees, including 55 that are known to be potentially more productive (APHP) and their 164 less productive immediate neighbours (non-APHP) in the four cardinal directions, over 15 administrative regions of the cashew production basin in Côte d'Ivoire (Figure 1). The leaves about 10-12 cm long and 6-8 cm wide, and the roots with a diameter of about 0.2-0.4 mm were collected during the dry season (February- March) and during the rainy season (July- August). These samples were stored in 70% alcohol before and during their preparation and observation under an optical microscope. Using a manual microtome and polystyrene, 0.1-0.2 mm microscopic sections of leaves, blade veins, midlines and roots were cut and stained using the classic green-carmino double staining technique (Ruzin 1999). To observe the sto- mata, a razor blade was placed on the lower surfaces of the leaves, and then the transparent epidermis located on the surfaces of the leaves was gently lifted. The fragments of the obtained transparent epidermis constituted the samples that were observed (Djinet et al. 2016). Anatomical tissues were visualized and recorded using Leica Acquire software. Image analysis The captured images were saved in 640×480 pixels format. Each anatomical section was measured with a micrometre graduated in millimetres (mm) and then converted to micrometres (µm) before observation under a microscope. Afterwards, the image of each anatomical section was measured in pixels at 10×, 20×, 40× and 100× magnifications. Therefore, calibration was done on each of the sections using this process to convert the photographs of the sections into µm. Histological analysis The anatomical tissues of leaves and roots were analyzed and measured with the Image-J 1.53t software. The param- eters taken into account were: • The thickness of the parenchymas, where the absorbed water is used for the photosynthetic activities; • the structure of the epidermis that regulates water exchange between the interior and the exterior of the cashew aerial organs; • the stomatal density and the surface of the opening of the ostiole of the stomata, which allow gas and water exchange between the interior and the exterior of the aerial organs of the cashew tree: • The characteristics of the xylems that conduct the absorbed water toward the cashew organs. Regarding the leaf midrib xylem diameter, the diagonal diameters were measured as recommended (Stein et al. 2017). The diameters of the root xylem vessels were measured taking into account both the tangential diameter (DT) and the radial diameter (DR); their ratio (R) was calcu- lated by the following expression: • The number of root xylem vessels was determined with the cell counter tool on 1 area of 1×1 mm (Grishaguin 1985). Statistical analysis The data collected in the Excel spreadsheet were analyzed using the R software to compare thicknesses and visualize them. The ANOVA test and Student t-test were performed to compare the data during the rainy and the dry seasons at the 5% significance level (p < 0.05). Pearson’s correlation test was performed to assess the relation between the number of xylem vessels and the ratio of the tangential and radial diameters of the vessels of the roots. Results Leaf tissues Parenchymas The average thicknesses of the lacunar parenchyma (Figure 2) of both APHPs and non-APHPs were larger during the rainy season than during the dry season (Table 1, Figure 3). The season did not show any impact (P = 0.090) on the thickness of the APHPs’ lacunar parenchyma, while drought showed a high negative impact (P = 0.009) on the thickness of the non-APHPs’ lacunar parenchyma, which decreased from 37 ± 1.5 µm during the rainy season to 36 ± DS = Number of stomata Leaf surface R = DT DR 56 Acta Biologica Slovenica, 2025, 68 (1) 3.6 µm during the dry season (Table 1). The season did not show any impact (P > 0.105) on the thicknesses of the APHPs’ lower and upper palisade parenchyma, while drought showed a high positive impact (P < 0.028) on the thicknesses of the non-APHPs’ palisade parenchymas (Table 1) which increased from 54 ± 4.4 µm during the rainy season to 59 ± 5.4 µm during the dry season at the upper face, and from 21 ± 10.9 µm during the rainy season to 22 ± 5.2 µm during the dry season at the lower face (Table 1, Figure 3). Figure 2. Pictures of the flattened blade of the cashew leaves according to the seasons (100× magnification). A: dry season; B: rainy season; PP: palisade parenchyma; LP: lacunar parenchyma; IS: intercellular space Slika 2. Slike sploščene listne ploskve indijskega oreščka glede na letni čas (100-kratna povečava). A: suha sezona; B: deževna sezona; PP: palisadni parenhim; LP: lakunarni parenhim; IS: medcelični prostor Tissue location Flattened blade tissues Midrib tissues Tree categories Season type Upper palisade parenchyma (µm) Lower palisade parenchyma (µm) Lacunar parenchyma (µm) Upper epidermis (µm) Lower epidermis (µm) Upper cortical parenchyma (µm) Lower cortical parenchyma (µm) Medullary parenchyma (µm2) Xylem bundles (µm) APHP Rainy season 52 ± 4.2a 20 ± 2.9a 40 ± 4.0a 5.6 ± 0.4a 5 ± 0.4a 8 ± 2.3a 18.5 ± 4.5a 4300 ± 837a 6.5± 0.7a Dry season 52 ± 6.3a 28 ± 2.1a 30± 6.3a 6.2 ± 1.0b 5.6 ± 1.1b 6.3 ± 0.8a 22 ± 3.4a 5800± 412a 6.4± 0.5b P-value 0.500 0.105 0.090 0.032 0.036 0.075 0.055 0.094 0.005 Non-APHP Rainy season 54 ± 4.4a 21 ± 10.9a 37 ± 1.5a 6.4 ± 0.3a 5 ± 0.4a 7.2 ± 1.2a 15 ± 4.9a 3400 ± 809a 6.7 ± 0.9a Dry season 59 ± 5.4b 22± 5.2b 36± 3.6b 6.5 ± 1.1b 5.5 ± 0.7b 6.2± 0.5b 20 ± 2.0a 5200 ± 993a 5.9 ± 0.6b P-value 0.028 0.015 0.009 0.005 0.030 0.047 0.090 0.131 0.040 Table 1. Average thickness of leaf tissues according to the cashew tree categories and to the seasons. APHP: potentially higher-producing cashew trees. For the tissue, paired data with the same letter (P-value > 5%) are statistically similar, while data with different letters are different (5% > P-value > 1%) or highly different (1% > P-value > 0.1%). Tabela 1. Povprečna debelina listnih tkiv glede na kategorije indijskih dreves in letne čase. APHP: drevesa indijskega oreščka s potencialno višjo rodnostjo. Pri tkivu so si parni podatki z isto črko (P-vrednost > 5 %) statistično podobni, medtem ko so podatki z različnimi črkami različni (5 % > P-vrednost > 1 %) ali zelo različni (1 % > P-vrednost > 0,1 %). 57 Acta Biologica Slovenica, 2025, 68 (1) No impact (P > 0.055) of the season was found on the thicknesses of the APHPs’ midrib in both face cortical parenchymas and on the non-APHPs’ lower face cortical parenchyma thickness (P = 0.090), while drought showed a negative impact (P = 0.047) on the thickness of the non- APHPs’ upper face cortical parenchyma which decreased from 7.2 ± 1.2 µm during the rainy season to 6.2 ± 0.5 µm during the dry season (Table 1, Figure 4). The season did not show any impact (P > 0.093) on the area of the medullary parenchyma of both APHPs and non-APHPs, even if the average area of the medullary parenchyma of the APHPs was about 4,300 ± 837 µm² during the rainy season and 5,800 ± 412 µm² during the dry season, while that of the non-APHPs was set at 3,400 ± 809 µm² during the rainy season and at 5,200 ± 993 µm² during the dry season (Table 1, Figure 5). Figure 3. Thickness of the parenchyma of flattened leaf blades. IE: lower palisade parenchyma; SE: upper palisade parenchyma. Slika 3. Debelina parenhima sploščenih listnih lopatic. IE: spodnji palisadni parenhim; SE: zgornji palisadni parenhim. 58 Acta Biologica Slovenica, 2025, 68 (1) Figure 4. Thickness of cortical parenchyma. Slika 4. Debelina kortikalnega parenhima. Figure 5. Area of the medullary parenchyma. Slika 5. Območje medularnega parenhima. 59 Acta Biologica Slovenica, 2025, 68 (1) Epidermis Firstly, both APHPs and non-APHPs showed a similar thickness of the upper and the lower epidermis, and secondly, an upper epidermis thicker than the lower epi- dermis, independently of the seasons (Table 1, Figure 6). Moreover, both categories of cashew leaves showed an increase (P ≤ 0.036) in the thickness of both the lower and upper epidermis from the rainy season to the dry season (Table 1, Figure 6). Xylem Both categories of cashew leaves showed the same thick- ness of xylem vessels for each season but a variation of the thickness of xylem vessels according to the seasons (Table 1, Figures 7 and 8). Indeed, there was a decrease (P = 0.040) in the non-APHPs’ xylem vessel thickness which moved from 6.7 ± 0.9 µm during the rainy season to 5.9 ± 0.6 µm during the dry season, and a high decrease (P = 0.004) of the APHPs’ xylem vessel thickness which changed from 6.5 ± 0.7 µm during the rainy season to 6.4 ± 0.5 µm the dry season (Table 1, Figure 7). Stomatal density and opening surface The average stomatal density at the abaxial faces of the leaves was the same (P ≥ 0.365) for the APHPs and the non-APHPs during each season. However, the average stomatal density of APHPs was lower, with 155 ± 16.9 stomata/mm2 during the rainy season than those of 190.4 ±41.6 stomata/mm2 during the dry season (Figure 9). The non-APHPs also showed a lower average stomatal density of 147 ± 15.6 stomata/mm2 during the rainy season than those of 170.4 ± 20.8 stomata/mm2 during the dry season (Figure 9). In both APHP trees and non-APHP trees, water deficit affected (P < 0.018) the opening width of the stomata, reducing it from 59.8 µm to 23.0 µm and from 47.3 to 22.2 µm, respectively, during the dry season (Figure 10). During each season, the stomata opening width was the same (P > 0.552) for the APHPs and the non-APHPs, which was 35.5 µm during the rainy season (Figure 11) and 19.2 µm during the dry season (Figure 12). Figure 6. Thickness of the upper and lower leaf epidermis according to the season. Slika 6. Debelina zgornje in spodnje listne povrhnjice glede na sezono. 60 Acta Biologica Slovenica, 2025, 68 (1) Figure 7. Xylem vessel thicknesses of the leaf midrib. Slika 7. Debelina ksilemskih žil na sredini lista. Figure 8. The anatomical structure of the main vein of cashew leaves according to the season (40× magnification). A: during the rainy season (production); B: during the dry season (after production). Slika 8. Anatomska zgradba glavne žile listov indijskega oreščka glede na sezono (40-kratna povečava). A: v deževnem obdobju (proizvod- nja); B: v suhem obdobju (po proizvodnji). 61 Acta Biologica Slovenica, 2025, 68 (1) Figure 9. The stomatal density of cashew trees. For each locality of origin of the cashew trees, the letter V is used to distinguish the non- APHPs from the APHPs. Slika 9. Stomatalna gostota indijskih dreves. Za vsako lokacijo izvora indijskih dreves je uporabljena črka V za razlikovanje med drevesi, ki niso APHP, in drevesi, ki niso APHP. Figure 10. Variation in the surface area of cashew tree stomata ostioles depends on the seasons. For each locality of origin of the cashew trees, the letter V is used to distinguish the non-APHPs from the APHPs. Slika 10. Razlike v površini ostiol listnih rež indijskega drevesa glede na letni čas. Za vsako lokacijo izvora indijskih dreves je uporabljena črka V za razlikovanje med drevesi, ki niso APHP, in drevesi, ki niso APHP. 62 Acta Biologica Slovenica, 2025, 68 (1) Root tissues Xylems The root xylems were characterized by both their vessel number and diameter. The APHPs’ root xylem vessel number varied between 11 and 26 vessels/mm2, while that of the non-APHPs varied between 15 and 42 vessels/mm2 (Figure 13). ANOVA did not show (P = 0.051) any impact of the season on the number of xylem vessels in both APHP and non-APHP roots. The largest root xylem radial diameters, set between 16.62-18.62 µm and tangential diameters set between 13.02-16.92 µm, were found during the rainy season (Figure 14), while the smallest radial diameters, about 4.88 µm and tangential diameters about 3.48 µm were found during the dry season (Figure 15). However, no impact of the season (P = 0.263) was found on the root xylem vessel diameter of both APHPs and non-APHP cashew trees. No correlation (P > 0.380) was found between the number of xylem vessels and the ratio of the tangential and radial diameters of the vessels of the roots of both APHPs and non-APHPs. For each locality of origin of the cashew tree, the letter V is used to distinguish the non-APHPs from the APHPs. Figure 11. Surface areas of stomatal ostioles during the rainy season. A: APHP; B: non-APHP (100× magnification). Slika 11. Površine stomatalnih ostiol v deževnem obdobju. A: APHP; B: brez APHP (100-kratna povečava). Figure 12. Surface areas of stomatal ostioles during the dry season. A: APHP; B: non-APHP (100× magnification). Slika 12. Površina stomatalnih ostiolov v sušnem obdobju. A: APHP; B: brez APHP (povečava 100×). 63 Acta Biologica Slovenica, 2025, 68 (1) Figure 13. The number of xylem vessels in the cashew tree roots according to the cashew categories and the season. Slika 13. Število ksilemskih žil v koreninah indijskega drevesa glede na kategorije indijskega oreščka in sezono. Figure 14. Tangential and radial diameters of the xylem vessels of cashew trees during the rainy season. Slika 14. Tangencialni in radialni premeri ksilemskih žil indijskih dreves med deževno sezono. 64 Acta Biologica Slovenica, 2025, 68 (1) Figure 15. Tangential and radial diameters of the xylem vessels of cashew trees during the dry season. Slika 15. Tangencialni in radialni premeri ksilemskih žil indijskih dreves v sušnem obdobju. Figure 16. Thickness of the medullary parenchyma of the cashew tree roots. The letter V is used to distinguish the non-APHPs from the APHPs. Slika 16. Debelina medularnega parenhima korenin indijskega drevesa. Črka V je uporabljena za razlikovanje med ne-APHP in APHP. Medullar parenchyma The average thickness of the roots medullary parenchyma increased with water deficit (Figure 16). The APHPs and non-APHPs experienced average medullary parenchyma thicknesses of 70 µm and 62 µm, respectively, during the dry season (Figure 16), while these values were about 44 µm for the APHPs and 20 µm for the non-APHP (Figure 17). Independently of the season, the APHPs showed a larger (P = 0.003) average medullary parenchyma thickness of roots medullar parenchyma than the non-APHPs (Figures 16 and 17). 65 Acta Biologica Slovenica, 2025, 68 (1) Figure 17. Roots medullary parenchyma width during the dry season (A) and the rainy season (B). Slika 17. Širina medularnega parenhima korenin v sušnem obdobju (A) in deževnem obdobju (B). Discussion Plants exposed to water deficits undergo biochemical changes as well as various anatomical adaptations, enabling them to grow under stress conditions (Saeed et al. 2016). In this study, water deficit conditions during the dry season significantly increased the cortical parenchyma of the main vein from 18.5 ± 4.5 μm to 22 ± 3.4 μm. This may indicate that cashew trees adapt their anatomical structure to water-limited environments. As Boughalleb et al. (2014) demonstrated in another study on Astragalus gombo Coss. & Durieu ex Bunge subsp. gomboeformis (Pomel) Ott, various anatomical adaptations, including cortical and mesophyll parenchyma formation, occur in response to limited water availability. These features are suggested to contribute to maintaining water potential and storing energy during drought, which effectively enhances plant survival in arid environments. A significant increase was also observed in leaf ana- tomical characteristics, such as the thickness of the pali- sade parenchyma on both surfaces (upper surface: 54 ± 4.2 μm to 59 ± 5.5 μm, lower surface: 20 μm ± 2.9 μm to 28 ± 2.1 μm) and in the thickness of the medullary parenchyma (from 4300 ± 837.7 μm² to 5800 ± 412.7 μm²) of the main vein during the dry season. These findings do not align with Akram et al. (2016), who reported that water stress in radish cultivars significantly reduced the main vein parenchyma thickness. Previous studies have shown that water stress can considerably reduce leaf mesophyll thickness and main vein thickness in Triticum aestivum L. (Burnett et al. 2005) and Ctenanthe setosa (Roscoe) Eichler (Kutlu et al. 2009). Similarly, Bosabalidis and Kofidis (2002) demon- strated that severe stress negatively affects mesophyll and palisade parenchyma thickness. Our findings also revealed a decrease in cashew lacunar parenchyma, regardless of production potential (Figure 5). Similarly, Fernández et al. (1997) found that water deficit in Olea europaea L. reduces mesophyll intercellular spaces, causing morphological and anatomical modifications in leaves. The reduction in epidermal cell thickness is an adaptive strategy that contributes to leaf resistance against cellular collapse due to drought and controls transpiration at the cuticle level (Bosabalidis and Kofidis 2002). However, in the cashew leaves, the epidermal cell size increased from 5.6 ± 0.4 μm during the rainy season to 6.2 ± 1.0 μm during the dry season, thus reducing water loss as stomatal density increased (Figure 9). Ennajeh et al. (2010) also observed that the lacunar parenchyma thickness is higher under water deficit. This increased thickness during the dry season in olives is attributed to an enlargement of intercellular spaces, which enhances CO₂ diffusion. The larger thickness of the xylem in the main leaf vein during the rainy season (6.7 ± 0.9 μm) than in the dry season (5.9 ± 0.6 μm) can likely be due to greater water availability in the soil. This water availability increases hydraulic conduc- 66 Acta Biologica Slovenica, 2025, 68 (1) tivity, which increases xylem thickness and vascular bundle number. Similar results were found in roots, with both vessel number (19.7 ± 3.9 μm to 27.8 ± 7.1 μm) and vessel thickness (radial diameter: 16.6-18.6 μm to 4.9 μm; tangential diameter: 13.0-16.9 μm to 3.5 μm) increasing during the rainy season. This strategy is also adopted by grapevine cultivars, which respond to water limitations by adopting specific hydraulic strategies (Lovisolo et al. 2010; Dal Santo et al. 2016). Moreover, the formation of smaller vessels under drought conditions has been observed in the stems of various tree species, such as Quercus L. and Populus L. (Arend and Fromm 2007, Fonti et al. 2013). Small vessels are suggested to be less vulnerable to xylem embolism induced by drought and contribute to water flow regulation under water-limited growth conditions. Additionally, the anatomical response to water limitation in grapevines includes adaptation through modification of xylem traits (xylem conduit size and number). Conversely, the increase in xylem vessel number and thickness enhances cavitation tolerance, conferring drought resistance (Arend and Fromm 2007). Smaller vessel diameters facilitate hydraulic conductance, reduce cavi- tation, and make water and nutrient uptake more efficient (Queiroz-Voltan et al., 2014). Under water stress, smaller xylem vessels promote efficient and safer water transport, improving hydraulic conductivity (Batista et al. 2010). In contrast, larger xylem vessels are more common in areas with higher rainfall for a given species (Mauseth 1988). The olive tree, a drought-resistant species, reduces xylem vessel diameter under water stress, allowing the plant to maintain transpiration flux (Ben et al. 2007). Our findings indicate that stomatal density in cashew trees ranges from 125 to 259 stomata per mm² (Figure 9). These results align with Esau (1965), who reported a den- sity range of 100-300 stomata per mm² in numerous plant species. Stomata are denser during the dry season (259 stomata/mm²) than the rainy season (176 stomata/mm²). The stomatal aperture is smaller in the dry season (23 μm) than in the rainy season (59.3 μm), consistent with Ma (2010), who found that increased osmotic pressure in guard cell vacuoles, following high solute absorption, induces stomatal pore opening. Raven et al. (2013) observed that under hot, dry conditions, plants enter water stress, trigger- ing the production of abscisic acid, a regulatory hormone secreted by leaves and roots that participates in various physiological regulation mechanisms. Under drought conditions, abscisic acid synthesis increases in the roots, where it is transported to the leaves to initiate prolonged stomatal closure (William 1995). Abscisic acid binds to its receptor in the plasma membrane, causing a calcium-medi- ated phosphorylation cascade that releases ions and leads to guard cell turgor loss, reducing stomatal opening. Thus, decreased stomatal aperture during osmotic stress is the most pronounced response (Munns and Tester 2008). In several fruit tree species, the response to water deficit involves reducing stomatal area, as seen in pistachio trees (Abbaspour et al. 2012), other Mediterranean species (Ben Ahmed et al. 2008), and the Olea europaea L. as found in Guerfel et al. (2009). This mechanism limits CO₂ assimilation during water deficits. Fernández et al. (1997) also observed that reduced CO₂ assimilation may decrease leaf mesophyll conductance. Moreover, the highest stomatal densities were observed in cashew trees during the dry season. Grisi et al. (2008) reported similar results for irrigated and non-irrigated coffee plants, with non-irrigated plants showing higher stomatal density. Similarly, Batista et al. (2010) showed that higher stomatal densities observed under water deficit in coffee plants indicate greater drought tolerance. Plants, being sessile, must adapt to various environmental factors, with stomata playing a crucial role in this function (Casson and Gray 2008). This supports the existence of drought resistance characteristics in non-irrigated coffee plants with higher stomatal density. Conclusion The conditions impacted leaf and root tissues. The thickness of the palisade parenchyma of non-APHPs remained con- stant, while that of APHPs increased considerably from the rainy season to the dry season. The two types of trees had the same reaction to water deficit, which was the increase in upper and lower epidermis thickness in order to reduce water loss during the dry season. During the dry season, the thickness of the xylem decreased, the average thickness of the cortical parenchyma of the lower midrib increased, and the average thickness of the cortical parenchyma of the lower midrib decreased, the thickness of the intercellular spaces of the lacunous parenchyma decreased, and the thickness of the medullary parenchyma of the midrib of the leaves of APHPs and their neighbours increased. The stomatal density increased during the dry season in all the cashew trees. However, the opening surface of the stoma- tal ostioles decreased during the dry season. These results 67 Acta Biologica Slovenica, 2025, 68 (1) showed the different anatomical adaptation strategies of the cashew tree to water constraints. Authors’ contributions Conceptualization, K.A.R.F., K.M.L., and K.N.F.; methodol- ogy, K.A.R.F., K.M.L., and K.N.F.; software, K.A.R.F.; valida- tion, K.M.L., and K.N.F.; formal analysis, K.A.R.F.; investiga- tion, K.A.R.F. ; resources, K.A.R.F. ; data curation, K.A.R.F. ; writing—original draft preparation, K.A.R.F. ; writing—review, and editing, K.M.L., and K.N.F.; visualization, K.A.R.F. , and K.M.L.; supervision, K.N.F.; project administration, K.N.F.; funding acquisition, K.N.F.. All authors have read, and agreed to the published version of the manuscript. Acknowledgments We are grateful to the owners of the cashew orchards, where the filed data were collected. We thank the tech- nicians of the Laboratory of Functional Genomics and Genetic Improvement at the Nangui Abrogoua University that hosted the anatomic preparations and observations for their help. We are grateful to Cashew National Research Programs who granted this work. Funding This work was supported by the Inter-Professional Research Fund, the Agricultural Council and the Cotton and Cashew Council (CCA) and their collaborators, which is the Project to Promote the Competitiveness of the Cashew Value Chain (PPCA), in the framework of which this study was funded. Data availability The authors confirm that the datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Conflicts of interest No potential conflict of interest was reported by the authors. References Abbaspour, H., Afshari, H., Abdel-Wahhab, A., 2012. Influence of salt stress on growth, pigments, soluble sugars and ion accumulation in three pistachio cultivars. In: J. Med. Plant. Res., 6, 2468-2473. https://doi.org/10.5897/JMPR11.1710 Akram, N.A., Shafiq, S., Ashraf, M., Aisha, R., Sajid, M.A., 2016. 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Plant Sci., 7, 1-16. https://doi.org/10.3389/fpls.2016.02047 69 1 ZRC SAZU, Jovan Hadži Institute of Biology, Novi trg 2, 1000 Ljubljana, Slovenia 2 University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana, Slovenia 3 Tržaška cesta 62, 1111 Ljubljana 4 University of Maribor, Faculty of Natural Sciences and Mathematics, Maribor, Slovenia * Corresponding author: All authors contributed equally to this work. For correspondence, see the individual chapters. Editors: Simona Strgulc Krajšek 2 and Tina Klenovšek 4 Citation: Jakob, A., Štampar, L., Lobnik Cimerman, Ž., Strgulc Krajšek, S., Vittori, M., Dolničar, D., (2024). Significant records of plants, algae, fungi, and animals in SE Europe and adjacent regions 3. Acta Biologica Slovenica 68 (1) Published: 23.12.2024 https://doi.org/10.14720/abs.68.01.21460 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Significant records of plants, algae, fungi, and animals in SE Europe and adjacent regions, 3 Aljaž Jakob 1, Laura Štampar 2, Žan Lobnik Cimerman 2, Simona Strgulc Krajšek 2, Miloš Vittori 2, Dren Dolničar 3 Abstract In this paper, we present two significant records of bryophytes in Slovenia, Cryphaea heteromalla and Riccia cavernosa, which were found in new phytogeographical regions. We report the three new records of an isopod Armadillo officinalis, a species that has been considered possibly extinct in Slovenia, and a new record of bat, Plecotus macrobullaris, from the Julian Alps. Keywords Cryphaea heteromalla; Riccia cavernosa; bryophytes; mosses, liverworts, flora; Armadillo officinalis; Isopoda; isopods; Plecotus macrobullaris; Chiroptera; bats; fauna; Slovenia 70 Acta Biologica Slovenica, 2025, 68 (1) Cryphaea heteromalla was hitherto known only from submediterranean parts in the southwestern part of Slovenia (Martinčič 2024). The localities are in Vipava Valley (Martinčič, 2016), from the area of Škocjan Caves (Strgulc Krajšek et al., 2023) and from the flysch Šavrini hills southeast of Koper, where the species is not rare in humid microlocalities (pers. obs.). The new record is the first find of this Atlantic-Mediterranean species (Hill et al., 2007) in the central part of Slovenia in the pre-Alpine phytogeographical region. It has been noted this species is benefiting and spreading after the increase in air quality in Europe towards the end of 20th and beginning of 21st century. However, in Germany, the spread of this species has additionally been linked to climate change and eutrophication of the environment (Müller, 2016). Since the new locality is in a highly anthropogenic environ- ment in intensely managed agricultural land, eutrophication and climate change might likely play a role in the spread of this species to continental parts of Slovenia. The species is expected to be found elsewhere in similar anthropogenic and eutrophic environments. Cryphaea heteromalla is very distinctive when fertile, it has erect sporophyte-bearing shoots with several capsules growing along just one side (Figure 1). The capsules are 2.5–3 mm long and have a very short seta (Preston, 2010). In Europe, confu- sion is only possible with larger Dendrocryphaea lamyana, which grows in regularly flooded habitats, such as river rocks and bases of riparian trees, and has blunter leaves (Preston, 2010; Casas, 2006). It is a rare plant with strictly Atlantic-Mediterra- nean distribution, and it is not expected to occur in Slovenia (Hill et al., 2007). Funding This research was funded by Slovenian Research and Innovation Agency (ARIS), grant numbers MR-55879 and P1-0236. Author(s) Aljaž Jakob Corresponding author Aljaž Jakob (aljaz.jakob@zrc-sazu.si) Leg. Aljaž Jakob Country Slovenia Statement of significance First record in Slovenia outside the submediterranean region Locality description Gorenjska, Medvode, Gorenja vas-Reteče, the gravel pit 1.2 km northeast of the village. Habitat In a partially flooded gravel pit, a semi-permanently flooded riparian forest, on Salix alba bark Date of observation 2024-06-28 Geographical coordinates N 46.16747309°, E 14.38683831° Voucher Photo documented Cryphaea heteromalla (Hedw.) D. Mohr., Crypheaceae (moss, bryophyte) 71 Acta Biologica Slovenica, 2025, 68 (1) Riccia cavernosa (Hoffm.) Hoffm. is a temperate species of a complex thalloid liverwort with a circumpolar distribution. It is also present in the tropics and the Southern Hemisphere (Hill & Preston, 1998). It is typically found in temporarily wet habitats such as the banks of ponds, ditches, rivers, wetlands, and other periodically inundated sites (Frey et al., 2006). R. cavernosa is a species from subgenus subgenus Ricciella (A. Braun) Boulay, section Spongodes Nees (Hodgetts et al., 2020). It is characterised by very distinctly perforated, yellow-green thallus without reddish colouration. A short, inconspicu- ous groove is visible on the upper surface only at the tip of the lobes (Long, 2010). The species has significant morphological similarities to R. crystallina L., therefore ripe spores are needed for reliable identification (Paton, 1999). Author(s) Laura Štampar, Žan Lobnik Cimerman, Simona Strgulc Krajšek Corresponding author Simona Strgulc Krajšek (simona.strgulc@bf.uni-lj.si) Leg. Laura Štampar Country Slovenia Statement of significance The third known locality in Slovenia, first in the dinaric phytogeographical region Locality description Notranjska, Cerkniško Jezero, Laze pri Gornjem Jezeru, left bank of river Jezerščica, next to the bridge near the Malnšče observation point, 550 m a.s.l. Habitat damp sandy soil Date of observation 2024-08-30 Geographical coordinates N 45.728611°, E 14.404167° Voucher Herbarium LJU (s.n.) Riccia cavernosa (Hoffm.) Hoffm., fam. Ricciaceae (liverwort, bryophyte) Figure 1. Cryphaea heteromala with characteristic erect shoots with several laterally growing capsules (photo: A. Jakob). 72 Acta Biologica Slovenica, 2025, 68 (1) R. cavernosa is listed as the least concerned (LC) in the European Red List (Hodgetts et al., 2019). It is widespread in Europe but rare in the Mediterranean region (Blockeel et al., 2014), and its conservation status varies significantly across different countries. In Italy and Austria, it is endangered (EN), in Hungary, it is near threatened (NT), and in Croatia, it has no conser- vation status (Hodgetts and Lockhart, 2020). In Slovenia, it was found for the first time in 2021 in the vicinity of Maribor by Ž. Lobnik Cimerman (Ellis et al., 2022) and later also near Dokleževje in the Prekmurje Region (Lobnik Cimerman et al., 2023). In Slovenia, it has a status of endangered species (Martinčič, 2024). Our sample was collected from a sandy substrate on the left bank of the Jezerščica River, approximately 80 meters down- stream from the bridge near the Malnšče observation point. The Jezerščica River is part of the Stržen River, flowing near Gorenje Jezero, in the vicinity of intermittent Cerknica Lake. The Stržen is one of the main streams of the lake, flowing through this karst region and frequently forming seasonal floodplains. The banks of the Stržen River host numerous other plant species, mainly amphibious macrophytes, due to the constant water level fluctuation, creating a dynamic environment ideal for their growth. Some of the species of amphibious macrophytes with the highest abundance, which had already been recorded by Kržič et al. (2007), and were confirmed by our observations, are Myosotis scorpioides, Nuphar luteum, Ranun- culus trichophyllus, Rorippa amphibia and Schoenoplectus lacustris. Riccia cavernosa appeared in considerable abundance at the locality, covering entire sections of the riverbank (Figure 2), which is characteristic of the species when water level drops and its ecological requirements are met. Some field obser- vations suggest that its spores can remain viable for several years (Blockeel et al., 2014). It appears that the current condi- tions are favourable for its growth, as the species is well adapted to fluctuations in water levels, typical of the area around Cerknica Lake and the Stržen River. However, excessive overgrowth of the riverbank could potentially threaten R. cavernosa. Funding This work was supported by the Slovenian Research Agency (ARIS) through the programme group P1-0212 and ARIS Young research grant (Ž. Lobnik Cimerman). Figure 2. Riccia cavernosa growing on humid sandy soil (photo: L. Štampar). 73 Acta Biologica Slovenica, 2025, 68 (1) The pill bug Armadillo officinalis Duméril, 1816 was first recorded on the territory of Slovenia in Škocjan near Koper in July 1926 (Strouhal, 1929). This record was the basis for the inclusion of the species in checklists of the Slovenian terrestrial isopod fauna (Potočnik, 1989; Vittori et al., 2023), but the species was not recorded in Slovenia again for nearly a century. Investigations of the terrestrial isopod fauna of the Slovenian coast in the 1970s and 1980s did not detect A. officinalis (Potočnik, 1984). As a result, the species was first considered endangered (Potočnik, 1992), and later, as efforts to find it had failed, possibly extinct in Slovenia (Potočnik, 1996). Despite this, it was not assigned a red list status in the national Red list of Malacostraca (Ur. l. RS., 2002). The first recent observation was of a single isopod under a wooden board at the saltpans at Fontanigge in September 2023 (Figure 3A). Two days later, more than ten individuals were observed near the Walls of Piran, again under a wooden board. Six isopods found there were collected and preserved for further examination (Figure 3B). A third observation occurred at Fontanigge in March 2024. This time, more than ten individuals were observed under wooden pallets near the Museum of Salt Making (Figure 3C). Species of the genus Armadillo are capable of rolling into a ball, with the flat protopodites of the uropods completing the outline of the pleon, and the uropod exopodites reduced to small cones that insert dorsally (Figure 3B). The genus is further characterized by monospiracular lungs on all pleopod exopodites and by modifications of pereopods 4 and 5 that enable these isopods to stridulate (Schmalfuss, 1996; Taiti et al., 1998; Cividini et al., 2020). While several species of Armadillo have been described from the Eastern Mediterranean, North Africa, and the Iberian Peninsula, A. officinalis is the only widespread Mediterranean species (Schmalfuss, 2003). It can be distinguished on the basis of the shape of the uropod protopodites and the pleotelson, the smoothness of the tergites, and male characters, mainly the shape of pereopod 7 and the exopodite of pleopod 1 (Schmalfuss, 1996). Author(s) Miloš Vittori Corresponding author Miloš Vittori (milos.vittori@bf.uni-lj.si) Leg. (1,2) Miloš Vittori(3) Barbara Breznik, Barbora Ďurajková, Miloš Vittori Country Slovenia Statement of significance New records of a species considered possibly extinct in Slovenia Locality description (1) Sečovlje, Fontanigge, elevation 2 m.a.s.l. (2) Piran, by the Walls of Piran, elevation 35 m.a.s.l. (3) Sečovlje, Fontanigge, by the Museum of Salt Making, elevation 2 m.a.s.l. Habitat (1) Mediterranean halophilous scrub (2) Paved street (3) Mediterranean halophilous scrub Date of observation (1) 2023-09-21 (2) 2023-09-23 (3) 2024-03-30 Geographical coordinates (1) N 45.47202°, E 13.6034° (2) N 45.52688°, E 13.57058° (3) N 45.47636°, E 13.59347° Voucher Six specimens collected in Piran are kept in the isopod collection of Miloš Vittori, University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana, Slovenia. Individuals observed at Fontanigge were not collected and were photographed instead. Armadillo officinalis Duméril, 1816, fam. Armadillidae (animal) 74 Acta Biologica Slovenica, 2025, 68 (1) The consistent presence of these isopods suggests that the population of A. officinalis in Slovenia may be recovering, with climate change possibly contributing to its success. Sket (2003) considered the species to be expanding its range, aided by human activity. Nevertheless, its recorded distribution in the country remains limited to the immediate proximity of the coast. Acknowledgement This work was supported by University Infrastructural Centre “Microscopy of Biological Samples” at the Biotechnical Faculty, University of Ljubljana. Funding This research received no external funding. Figure 3. Armadillo officinalis. A: The individual observed at Fontanigge in September 2023. B: One of the specimens collected in Piran in September 2023. The uropod protopodites complete the outline of the pleon and the uropod exopodites are diminutive (arrow- head). C: Individuals photographed at Fontanigge in March 2024. (photos A, B, C: Miloš Vittori). 75 Acta Biologica Slovenica, 2025, 68 (1) On 11 July 2024, I found 2 individuals of Plecotus in the space between the planks and beams of the barn (WGS 84: 46.287972° N, 13.763278° E; Figure 4) next to the mountain hut Koča pod Bogatinom (address: Ukanc 148), on the Komna plateau, at 1515 m a.s.l. One was too far away to determine the species, while the other one, which was closer, I was able to identify as Plecotus macrobullaris based on the characteristic inverted triangular shape of the lower lip (Dietz & Kiefer, 2016). The bat, identified as P. macrobullaris, was initially positioned much closer to the edge of the gap between the barn’s planks (Figure 4B), with its dark greyish triangular lower lip (Dietz & Kiefer, 2016) clearly visible. However, it retreated deeper into the gap after we disturbed it with light while attempting to photograph it. The record is the highest roost of this species in Slovenia, since the previously highest elevation known roost was in the church sv. Duha in Podolševa at 1250 m a.s.l. (Presetnik et al., 2023). It is the third highest observation overall, as the highest published record originates from the mist netting at Planina pri Jezeru at 1750 m a.s.l. (Alberdi et al., 2013; CKFF, 2024) and the second highest is an ultrasound recording from Planina Krstenica at 1670 m a.s.l. (A. Zamolo, personal communication, August 28, 2024). According to the general species vertical distribution, the finding of P. macrobularis at this altitude on Komna is not surprising. The species is common in the Alpine regions and is found in all the main mountain ranges of the western Palearctic, stretching from the Pyrenees to the Middle East, and is distributed at altitudes ranging from sea level to 2800 m a.s.l. (Alberdi et al., 2013). The scarcity of observations of P. macrobullaris in Slovene montane belt (CKFF, 2024) therefore indicates a lack of research in the Slovenian high mountains. Acknowledgement I would like to thank Primož Presetnik for gathering and sharing the relevant data about P. macrobullaris in Slovenia and for reviewing the first draft of this article and Aja Zamolo for sharing her records of this species. I would also like to thank Simona Strgulc Krajšek for sharing her photograph of P. macrobullaris that she took when I found it in the barn. Funding This research received no external funding. Author(s) Dren Dolničar Corresponding author Dren Dolničar (dren.dolnicar@gmail.com) Leg. Dren Dolničar Country Slovenia Statement of significance The highest elevation of recorded roost site for this species in Slovenia. Locality description Triglav National Park, Komna plateau, 1515 m a.s.l. Habitat An alpine meadow below the tree line, with a roost site in a wooden barn. Date of observation 2024-07-11 Geographical coordinates N 46.287972°, E 13.763278° Voucher / Plecotus macrobullaris Kuzyakin, 1965 (Chiroptera, Mammalia) (bats, mammals) 76 Acta Biologica Slovenica, 2025, 68 (1) Figure 2. A) Plecotus macrobullaris; B) the roosting place; C) the barn near the hut Koča pod Bogatinom on Komna, where at least two Ple- cotus bats were roosting (photo: A: Simona Strgulc Krajšek; B, C: Dren Dolničar). A B C References Alberdi, A., Garin, I., Aizpurua, O., Aihartza, J., 2013. Review on the Geographic and Elevational Distribution of the Mountain Long-Eared Bat Plecotus macrobullaris, Completed by Utilising a Specific Mist-Netting Technique. Acta Chiropterologica 15(2): 451–461. https://doi.org/10.3161/150811013X679071. Blockeel, T. L., Bosanquet, S. D. S., Hill, M. O., Preston, C. D. (eds.), 2014. Atlas of British and Irish bryophytes: the distribution and habitat of mosses and liverworts in Britain and Ireland. 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Acta Biologica Slovenica 68 (1) Received: 17.09.2024 / Accepted: 19.12.2024 / Published: 20.12.2024 https://doi.org/10.14720/abs.68.01.19821 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Cellulase, xylanase, lipase, and protease activities of selected wild mushrooms from Slovenia Larisa Lara Popošek 1, Luka Šparl 2, Ivona Pleše 1, Matija Hrovatin 1, Drejc Flajnik 1, Kristina Sepčić 1, Matej Skočaj 1* Abstract There are more than 3000 species of wild mushrooms in Slovenia. These wild mushrooms represent a promising source of bioactive compounds that can also be used as a source of novel enzymes, thus offering potential economic benefits for a wide range of industries and medicine. In this work, the cellulase, xylanase, protease, and lipase activities were measured in aqueous extracts of 64 Slovenian wild mushrooms of different lifestyles (symbiotic, saprobic, parasitic). We have shown that many of the mushroom extracts tested contain a variety of specific hydrolases. Of the 64 wild mushroom extracts, 16 showed cellulase, 24 xylanase, 30 lipase, and five protease activities. These are the first reports of these enzymatic activities for some of the mushrooms tested. Keywords mushroom; enzyme; cellulase; xylanase; lipase; protease 79 Acta Biologica Slovenica, 2025, 68 (1) Celulazne, ksilanazne, lipazne in proteazne aktivnosti nekaterih samoniklih slovenskih gob Izvleček V Sloveniji je opisanih več kot 3000 gob, ki predstavljajo potencialni vir bioaktivnih molekul kot so biotehnološko uporabni encimi, ki bi se jih lahko uporabilo v različnih industrijskih procesih ali v medicini. V sklopu raziskave smo pripravili vodne izvlečke 64 različnih slovenskih samoniklih gob, ki smo jim določili celulazno, ksilanazno, proteazno in lipazno aktivnost. Pokazali smo, da ti vodni izvlečki vsebujejo različne kombinacije encimskih aktivnosti. V vodnih izvlečkih teh 64 različnih slovenskih gob smo pokazali, da ima 16 izvlečkov celulazno, 24 ksilanazno, 30 lipazno in pet proteazno aktivnost. Mnoge od teh aktivnosti v literaturi še niso bile opisane. Ključne besede gobe, encim, celulaze, ksilanaze, lipaze, proteaze Introduction The global market for industrial enzymes was estimated at USD 60.5 billion in 2023 and is expected to grow by 4.9 % annually from 2024 to 2030 (Enzymes Market Size, Share, Trends and Growth Report, 2030). Approximately 60 % of industrial enzymes are produced by fungi (Raveendran et al., 2018). Many of these enzymes are used in various indus- tries, e.g. in the food, detergent, paper, and textile indus- tries, as well as in medicine (Pandey et al., 2000). Proteases, which account for 60 % of industrial enzymes, catalyse the depolymerization of proteins to amino acids and are used in the detergent, pharmaceutical, and food industries (Singh et al., 2016; Raveendran et al., 2018). Lipases are enzymes that hydrolyse triglycerides into fatty acids and glycerol. They are indispensable for the production of food, biofuels, deter- gents, and animal feed (Raveendran et al., 2018). Cellulases are a group of hydrolases that act on the β-1,4-glycoside bond in cellulose to release glucose units (Raveendran et al., 2018). This class of enzymes is widely used in the pro- duction of food as well as in the textile, paper, and detergent industries (Sukumaran et al., 2005). Xylanases are a group of enzymes that cleave xylenes, which are a major compo- nent of the plant cell wall and are mainly used in the food industry (Raveendran et al., 2018). Although cellulases and xylanases have no pharmacological potential, this is not the case for lipases and proteases. Lipases are being actively investigated for the treatment of modern diseases such as obesity or even cancer (Jawed et al., 2019). Proteases, on the other hand, can be used to combat cardiovascular dis- eases, digestive disorders, and inflammatory diseases and to promote tissue repair in burns, bone fractures, or surgical trauma (Solanki et al., 2021). Basidiomycota and Ascomycota are the major phyla of the fungal kingdom, and many of them produce spore-bear- ing fruiting bodies known as mushrooms (Schmidt-Dannert, 2016). As mushrooms can grow on a variety of substrates and have versatile lifestyles (symbiotic, saprobic, parasitic), they have evolved a powerful enzymatic mechanism. In this regard, mushrooms represent a promising source of novel and undiscovered enzymes that could offer great eco- nomic benefits to the industry. We, therefore, collected 65 wild mushrooms and determined their cellulase, xylanase, protease, and lipase activities. Materials and Methods Mushroom extract preparation The mushrooms were collected in Slovenia (Table 1) between December 2019 and February 2021, identified to species level, cut into small pieces and frozen at -80 °C. To obtain aqueous mushroom extracts, the thawed fruiting bodies were homogenized in 50 mM TRIS-HCl buffer, pH 7.4. For 1 g of wet biomass, 1 mL of the pre-cooled buffer was used, except for mushrooms growing on wood, which absorbed more buffer. We mixed 2 mL of the pre-cooled buffer per 1 g of such wet mushrooms. Pieces of the thawed mushroom fruiting bodies were then homogenized in the 80 Acta Biologica Slovenica, 2025, 68 (1) Waring Blender (Ika, Germany) for 10 seconds at maximal speed. Mushroom samples weighing less than 1 g were first frozen with liquid N2 and then homogenized in a mortar. Fruiting bodies that were homogenized using either a blender or a mortar were additionally homogenized using the Potter-Elvehjem homogenizer (Ika, Germany). Ten passages at 180 rpm were required to ensure complete homogenization of the mushroom biomass. The homog- enized fungal biomass was then centrifuged at 4 °C and 11,900 × g for 30 min. 1 mL of the clear supernatant was then pipetted into a 1.5 mL centrifuge tube and frozen at -20 °C. Commercial kits The BCA assay (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify total proteins, the Megazyme endo- cellulase assay and the Megazyme endoxylanase assay (Megazyme, Bray, Ireland) were used to measure cellulase and xylanase activities, and the Protease Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure total protease activity. Cellulase, xylanase, and protease activities were determined by the protein concentration in the sample, which was the enzyme unit (EU)/mg protein. Lipase activity To determine the lipase activity, 50 mM 4-nitrophenyl butyrate (p-NPB) was prepared in acetonitrile as a substrate. After cleavage of the ester bonds in the substrate, a yellow product 4-nitrophenol is formed, which absorbs at 450 nm. For the lipase reaction, a lipase buffer containing 1 ml Triton X-100, 100 mM sodium phosphate and 150 mM NaCl, pH 7.2, was prepared. The aqueous mushroom extracts were thawed and incubated on ice before the experiment. First, 5 μl p-NPB and 450 μl lipase buffer were mixed and incu- bated at 37 °C for 10 min. Aqueous mushroom extract (45 µl) was then added, and after 10 min of incubation at 37 °C, the reaction was stopped by adding acetone (750 µl). The Eppendorf tubes were then centrifuged for 5 min at room temperature at 15,000 × g, and 200 µl of the supernatant was pipetted into the wells of a 96-well microtiter plate. For the negative control, 5 µl of lipase buffer was added instead of 5 µl of p-NPB. An additional negative control, which was prepared due to the strong absorption of p-NPB, consisted of 495 µl lipase buffer and 5 µl p-NPB. After incubation of negative control samples at 37 °C for 10 min, 750 µl acetone was added, and the mixture was centrifuged at room tem- perature for 5 min at 15,000 × g. 200 µl of the supernatant was pipetted into microtiter plates. The absorbance was measured using the VIS microplate reader (Dynex Tech- nologies, Chantilly, VA, USA) at a wavelength of 450 nm. The test was repeated three times for each positive sample. Lipase activity was determined by the protein concentration in the sample as enzyme unit (EU)/mg protein. An EU is defined as the amount of sample that causes a change in absorbance at 450 nm of 1 mOD in one minute. Order Family Species Bionomial name Lifestyle1 Edibility2 Agaricales Hygrophoraceae Hygrocybe conica Hygrocybe conica (Schaeff.) P. Kumm. 1871 a* f Hygrocybe coccinea Hygrocybe coccinea (Schaeff.) P. Kumm. 1871 a* f Hygrocybe ovina Neohygrocybe ovina (Bull.) Herink 1958 a* d Hygrocybe citrinovirens Hygrocybe citrinovirens (J.E. Lange) Jul. Schäff. 1947 a* f Cuphophyllus pratensis Cuphophyllus pratensis (Pers.) Bon 1985 a* e Cuphophyllus fornicatus Cuphophyllus fornicatus (Fr.) Lodge, Padamsee & Vizzini 2013 a* f Gliophorus laetus Gliophorus laetus (Pers.) Herink 1958 a* f Porpolomopsis calyptriformis Porpolomopsis calyptriformis (Berk.) Bresinsky 2008 a* e Table 1. Wild mushrooms were collected within this study. Tabela 1. Divje gobe, zbrane v tej študiji. 81 Acta Biologica Slovenica, 2025, 68 (1) Tricholomataceae Tricholoma stiparophyllum Tricholoma stiparophyllum (N. Lund) P. Karst. 1879 b f Lepista nuda Lepista nuda (Bull.) Cooke 1871 c g Physalacriaceae Flammulina velutipes Flammulina velutipes (Curtis) Singer 1951 c e Pluteaceae Volvopluteus gloiocephalus Volvopluteus gloiocephalus (DC.) Vizzini, Contu & Justo 2011 c e Pleurotaceae Pleurotus ostreatus Pleurotus ostreatus (Jacq.) P. Kumm. 1871 a, c e Pleurotus pulmonarius Pleurotus pulmonarius (Fr.) Quél. 1872 a, c e Schizophyllaceae Schizophyllum commune Schizophyllum commune Fr. 1815 a, c f Marasmiaceae Megacollybia platyphylla Megacollybia platyphylla (Pers.) Kotl. & Pouzar 1972 c e Amanitaceae Amanita eliae Amanita eliae Quél. 1872 b d Amanita rubescens Amanita rubescens Pers. 1797 b g Amanita excelsa Amanita excelsa (Fr.) Bertill. 1866 b e Strophariaceae Stropharia eximia Stropharia eximia Benedix 1961 a e Hypholoma fasciculare Hypholoma fasciculare (Huds.) P. Kumm. 1871 a d Omphalotaceae Gymnopus dryophilus Gymnopus dryophilus (Bull.) Murrill 1916 a f Cortinariaceae Cortinarius bolaris Cortinarius bolaris (Pers.) Zawadzki 1835 b f Nidulariaceae Cyathus striatus Cyathus striatus Willd. 1787 c f Russulales Russulaceae Russula queletii Russula queletii Fr. 1872 b f Russula virescens Russula virescens (Schaeff.) Fr. 1836 b e Russula nigricans Russula adusta (Pers.) Fr. 1838 b e Russula amoenolens Russula amoenolens Romagn. 1952 b f Russula cyanoxantha Russula cyanoxantha (Schaeff.) Fr. 1863 b e Lactarius torminosus Lactarius torminosus (Schaeff.) Pers. 1797 b f Lactarius volemus Lactifluus volemus (Fr.) Kuntze 1891 b e Lactarius vellereus Lactifluus vellereus (Fr.) Kuntze 1891 b f Cantharellales Hydnaceae Hydnum repandum Hydnum repandum L. 1753 b e Cantharellaceae Cantharelus friesii Cantharellus friesii Quél. 1872 b e Cantharellus cibarius Cantharellus cibarius Fr. 1812 b e Cantharellus amethysteus Cantharellus amethysteus (Quél.) Sacc. 1887 b e Polyporales Fomitopsidaceae Fomitopsis betulina Fomitopsis betulina (Bull.) B.K. Cui, M.L. Han & Y.C. Dai 2016 a, c f 82 Acta Biologica Slovenica, 2025, 68 (1) Daedalea quercina Daedalea quercina (L.) Pers. 1801 a, c f Laetiporaceae Laetiporus sulfureus Laetiporus sulphureus (Bull.) Murrill 1920 a, c g Polyporaceae Daedaleopsis confragosa Daedaleopsis confragosa (Bolton) J. Schröt. 1888 c f Trametes hirsuta Trametes hirsuta (Wulfen) Lloyd 1924 c f Trametes versicolor Trametes versicolor (L.) Lloyd 1921 c f Trametes suaveolens Trametes suaveolens (L.) Fr. 1838 c f Trametes gibbosa Trametes gibbosa (Pers.) Fr. 1838 c f Fomes fomentarius Fomes fomentarius (L.) Fr. 1849 a, c f Neofavolus alveolaris Neofavolus alveolaris (DC.) Sotome & T. Hatt. 2012 c f Polyporus ciliatus Lentinus substrictus (Bolton) Zmitr. & Kovalenko 2016 c f Cerioporus leptocephalus Cerioporus leptocephalus (Jacq.) Zmitr. 2016 c f Meruliaceae Phlebia tremellosa Phlebia tremellosa (Schrad.) Nakasone & Burds. 1984 c f Irpicaceae Gloeoporus taxicola Meruliopsis taxicola (Pers.) Bondartsev 1959 c f Auriculariales Auriculariaceae Auricularia mesenterica Auricularia mesenterica (Dicks.) Pers. 1822 c f Auricularia auricula-judae Auricularia auricula-judae (Bull.) Quél. 1886 c e Melanommataceae Phragmotrichum chailletii Phragmotrichum chailletii Kunze 1823 c f Hymenochaetales Hymenochaetaceae Phylloporia ribis Phylloporia ribis (Schumach.) Ryvarden 1978 a, c f Boletales Boletaceae Boletus reticulatus Boletus reticulatus Schaeff. 1774 b e Strobilomyces strobilaceus Strobilomyces strobilaceus (Scop.) Berk. 1851 b f Tylopilus felleus Tylopilus felleus (Bull.) P. Karst. 1881 b f Tapinellaceae Tapinella atrotomentosa Tapinella atrotomentosa (Batsch) Šutara 1992 c f Sclerodermataceae Scleroderma citrinum Scleroderma citrinum Pers. 1801 b d Suillaceae Suillus luridiformis Neoboletus luridiformis (Rostk.) Gelardi, Simonini & Vizzini 2014 b e Gloeophyllales Gloeophyllaceae Gloeophyllum odoratum Gloeophyllum odoratum (Wulfen) Imazeki 1943 c f Helotiales Sclerotiniaceae Mitrula paludosa Mitrula paludosa Fr. 1816 c f Chlorociboriaceae Chlorociboria aeruginascens Chlorociboria aeruginascens (Nyl.) Kanouse 1948 c f Pezizales Tuberaceae Tuber aestivum Tuber aestivum (Wulfen) Spreng. 1827 b e 1 a*, parasitic biotrophs; a, parasitic; b, symbiotic; c, saprobic. 2 d, poisonous; e, edible; f, inedible; g, conditionally edible. 83 Acta Biologica Slovenica, 2025, 68 (1) Results Of the 64 wild mushrooms examined, 23 were symbiotic fungi, and 41 had a saprobic or/and parasitic lifestyle. None of the examined mushrooms showed all four enzymatic activities, and 17 mushrooms had no enzymatic activities. In 23 mushroom samples, we could detect one or two enzymatic activities. The mushroom extracts from Tricholoma stiparophyllum and Trametes suaveolens showed three different enzymatic activities. We also inves- tigated the correlations between the edibility (for humans) of the mushrooms and their enzymatic activities and found no correlations. Cellulase activity Sixteen of 64 wild mushrooms (25 %) showed cellulase activity (Figure 1), and 12 of these 16 mushrooms were not symbiotic. The highest cellulase activities were found in aqueous extracts of Fomitopsis betulina and Cyathus striatus. The mushrooms for which cellulase activity was already determined were Pleurotus ostreatus, Pleurotus pulmonarius, Cyathus striatus, Fomitopsis betulina, Trametes versicolor, Trametes suaveolens, Trametes gibbosa and Auricularia auricula-judae (Yerkes, 1967; Bhattacharjee et al., 1992; Valášková and Baldrian, 2006; Wang et al., 2014; Araújo et al., 2021; Beyisa Benti Diro et al., 2021; Montoya et al., 2021; Lu et al., 2022). Cellulase activity was determined for the first time for eight of these 16 wild mushrooms. These mushrooms were Gliophorus laetus, Porpolomopsis calyptriformis, Tricholoma stiparo- phyllum, Volvopluteus gloiocephalus, Cantharelus friesii, Cantharellus amethysteus, Phragmotrichum chailletii and Suillus luridiformis. Figure 1. Aqueous extracts from wild mushrooms with detected cellulase activity. The mean values of triplicate samples with standard errors are shown. In green: the mushrooms for which cellulase activities are reported for the first time. Slika 1. Vodni izvlečki divjih gob z ugotovljeno aktivnostjo celulaze. Prikazane so povprečne vrednosti trikratnih vzorcev s standardnimi napakami. V zeleni barvi: gobe, za katere so bile celulazne aktivnosti navedene prvič. 84 Acta Biologica Slovenica, 2025, 68 (1) Xylanase activity Twenty-four of 64 wild mushrooms (37.5 %) exhibited xylanase activity (Figure 2), of which 19 were non-symbi- otic. The highest xylanase activities were found in aque- ous extracts of Phragmotrichum chailletii and Stropharia eximia. The mushrooms for which xylanase activity has already been determined were Pleurotus pulmonarius, Schizophyllum commune, Hydnum repandum, Fomitopsis betulina, Trametes versicolor and Trametes gibbosa (Paice et al., 1978; Valášková and Baldrian, 2006; Inácio et al., 2015; Megersa and Alemu, 2019; Sergentani et al., 2016; Tišma et al., 2021). For 19 of these 24 wild mushrooms, we have demonstrated their xylanase activity for the first time. These mushrooms were Hygrocybe ovina, Hygrocybe citrinovirens, Lepista nuda, Volvopluteus gloiocephalus, Amanita eliae, Stropharia eximia, Gymnopus dryophilus, Cyathus striatus, Russula nigricans, Cantharelus friesii, Trametes suaveolens, Neofavolus alveolaris, Polyporus ciliatus, Phlebia tremellosa, Gloeoporus taxicola, Phrag- motrichum chailletii, Tylopilus felleus, and Chlorociboria aeruginascens. The combined results of cellulase and xylanase activities of these wild mushrooms indicate that these two activities are more pronounced in non-symbiotic mushrooms. Lipase activity Our results show that of the four enzymatic activities tested, lipase activity is the most prevalent in the mushroom sam- ples. Thirty of 64 wild mushrooms (46.9%) exhibited lipase activity (Figure 3), and it was somehow more pronounced in non-symbiotic mushrooms, as 19 of 30 mushrooms were non-symbiotic. Flammulina velutipes, Daedaleopsis Figure 2. Aqueous extracts from wild mushrooms with detected xylanase activity. The mean values of triplicate samples with standard errors are shown. In green: the mushrooms for which xylanase activities were reported for the first time. Slika 2. Vodni izvlečki divjih gob z ugotovljeno ksilanazno aktivnostjo. Prikazane so povprečne vrednosti trikratnih vzorcev s standardnimi napakami. V zeleni barvi: gobe, za katere so bile ksilanazne aktivnosti navedene prvič. 85 Acta Biologica Slovenica, 2025, 68 (1) confragosa, Cuphophyllus fornicatus, Polyporus ciliates and Lactarius torminosus had the highest lipase activities. The mushrooms for which lipase activity had already been determined were Schizophyllum commune, Amanita rubescens, Lactarius torminosus, Laetiporus sulfureus and Daedaleopsis confragosa (Jaya et al., 2009; Krupodorova et al., 2014; Singh et al., 2015; Sepčić et al., 2019). We have demonstrated their lipase activity for 24 of these 30 wild mushrooms for the first time. These mushrooms were Hygrocybe conica, Hygrocybe coccinea, Hygrocybe ovina, Cuphophyllus pratensis, Cuphophyllus fornicatus, Porpolo- mopsis calyptriformis, Tricholoma stiparophyllum, Lepista nuda, Flammulina velutipes, Megacollybia platyphylla, Amanita eliae, Stropharia eximia, Gymnopus dryophilus, Russula queletii, Russula virescens, Russula amoenolens, Lactarius volemus, Trametes suaveolens, Neofavolus alveolaris, Polyporus ciliatus, Phlebia tremellosa, Boletus reticulatus, Tylopilus felleus and Suillus luridiformis. In addi- tion, the five mushrooms that exhibited protease activity also showed lipase activity. Protease activity Five out of 64 wild mushrooms (7.8 %) showed protease activity (Figure 4). Four of the five mushrooms that showed protease activity were non-symbiotic fungi. The highest protease activity was found in the aqueous extract of Daedaleopsis confragosa. For all these five wild mush- rooms, we are the first to detect their protease activity. These mushrooms were Hygrocybe conica, Hygrocybe coccinea, Cuphophyllus pratensis, Tricholoma stiparophyl- lum and Daedaleopsis confragosa. Figure 3. Aqueous extracts from wild mushrooms with detected lipase activity. The mean values of triplicate samples with standard errors are shown. In green: the mushrooms for which lipase activities are reported for the first time. Slika 3. Vodni izvlečki divjih gob z ugotovljeno aktivnostjo lipaze. Prikazane so povprečne vrednosti trikratnih vzorcev s standardnimi napa- kami. V zeleni barvi: gobe, za katere so prvič poročali o aktivnosti lipaz. 86 Acta Biologica Slovenica, 2025, 68 (1) Discussion Microbial hydrolases, including those from fungi, are the most important source of industrial enzymes. Fungal-de- rived enzymes account for more than 50% of the total enzyme market, and most of these enzymes are derived from moulds (El-Gendi et al., 2021). Almost 50% of these enzymes are produced by recombinant host strains, and most of them are also moulds (Arnau et al., 2019). Moulds are preferred enzyme factories because they can be grown in relatively inexpensive substrates under submerged conditions (Arnau et al., 2019). Since these enzymes are secreted extracellularly by moulds, they are relatively easy to separate from the liquid culture media. However, this does not apply to saprobic mushrooms, which have to be cultivated on solid culture media. The extraction of indus- trial enzymes from moulds is, therefore, less time-consum- ing, labour-intensive and more economically efficient than the extraction of enzymes from substrates overgrown with mushroom mycelia. While saprobic mushrooms can at least be cultivated on solid culture media, this is not always possible with symbiotic mushrooms, as only a few of them can be cultivated under laboratory conditions. Alternative solutions for the production of mushroom enzymes should, therefore, be explored. One of the pos- sible solutions is the cloning of genes from the producing mushrooms into suitable host(s). For this purpose, genome sequencing of mushrooms with high enzymatic activity should be performed. The target genes should be anno- tated, cloned and expressed in suitable microbial or fungal hosts, and the activity of these recombinant enzymes should be evaluated. By using recombinant technology to produce these enzymes in a host organism, it is possible to obtain large quantities of enzymes with the desired prop- erties, which could lead to more efficient and sustainable industrial processes. In this context, Phragmotrichum chail- Figure 4. Aqueous extracts from wild mushrooms with detected protease activity. The mean values of triplicate samples with standard errors are shown. In green: the mushrooms for which protease activities are reported for the first time. Slika 4. Vodni izvlečki divjih gob z ugotovljeno proteazno aktivnostjo. Prikazane so povprečne vrednosti trikratnih vzorcev s standardnimi napakami. V zeleni barvi: gobe, za katere so proteazne aktivnosti navedene prvič. 87 Acta Biologica Slovenica, 2025, 68 (1) letii and Stropharia eximia are proving to be a source of potent xylanases, Flammulina velutipes a source of potent lipase and Daedaleopsis confragosa a source of potent lipase and protease. Although new fungal species have been identified in this work as producers of important industrial enzymes, it is not possible to compare these activities with the activ- ities of pure enzymes derived from moulds grown under laboratory conditions. This is because these industrial mould strains grow in inducible substrates that trigger higher production of the enzymes. While the industrial enzymes produced by moulds are extracted directly from the liquid medium in which the moulds are grown, the extracts used in our study were derived directly from the producing organism. Since most symbiotic mushrooms cannot grow under laboratory conditions, recombinant expression of the target genes is the only solution for the evaluation and possible production of these enzymes on an industrial scale. Conclusions A growing number of industrial processes are replacing chemical-based solutions with biological-based solutions, which require new and effective enzymes. Our study, therefore, focused on the search for enzymes in higher fungi growing in nature, namely those that form fruiting bodies, which we believe represent an underestimated source of previously unexplored industrially important enzymes. The purpose of our study was to determine the cellulase, xylanase, lipase, and protease activities of 64 mushrooms collected in nature. This is the first report on the ability of many of these mushrooms to produce these hydrolytic enzymes. The findings of this study provide valu- able insight into the occurrence of cellulases, xylanases, lipases, and proteases in wild mushrooms. They highlight, in particular, the diversity of mushrooms that can produce these enzymes. The use of recombinant technology to introduce genes encoding cellulase, xylanase, lipase and protease from mushrooms into a host organism may have various applications, particularly in industries such as bio- technology, agriculture, biofuel production and medicine. Author Contributions Conceptualization, M.S.; methodology, M.S. and K.S; val- idation, M.S.; formal analysis, I.P. and L.L.P; investigation, L.Š., L.L.P, I.P., M.H. and D.F.; resources, M.S. and K.S.; data curation, M.S.; writing—original draft preparation, M.S.; writ- ing—review and editing, M.S., L.Š., I.P., M.H., D.F. and K.S.; visualization, L.P.P.; supervision, M.S.; project administra- tion, M.S.; funding acquisition, M.S. All authors have read and agreed to the published version of the manuscript. Acknowledgement The authors thank the anonymous reviewers for helpful comments that improved the quality of the manuscript. Funding This research was funded by the Slovenian Research and Innovation Agency, grant number P1-0207. Data Availability All raw or analysed data are available from the correspond- ing author upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. 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Process for preparing a cellulolytic enzyme from Trametes suaveolens (United States Patent US3310476A). https://patents.google.com/ patent/US3310476A/en 89 1 Laboratory of Bioconversion, Microbiology Engineering and HealthSafety, Faculty SNV, University of Mustapha Stambouli - Mascara, Algeria 2 Laboratory of Physical and Chemistry of Macromolecules and Biological Interface University of Mustapha Stambouli - Mascara, Algeria * Corresponding author: E-mail address: abderrahmane.ghillace@univ-mascara.dz Citation: Abderrahmane, G., Ourida, C., Zohra, K. F., Zineb, F., Zineeddine D., (2024). Phytochemical composition and antimicrobial activity of essential oil and extracts from the leaves of Peganum harmala L. in southern Algeria. Acta Biologica Slovenica 68 (1) Received: 26.11.2024 / Accepted: 07.01.2025 / Published: 09.01.2025 https://doi.org/10.14720/abs.68.01.21296 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Phytochemical composition and antimicrobial activity of essential oil and extracts from the leaves of Peganum harmala L. in southern Algeria. Ghillace Abderrahmane 1*, Chouitah Ourida 1, Kiari Fatima Zohra 1, Fergoug Zineb 2, Daikh Zineeddine 2 Abstract Medicinal plants have a wide use in traditional medicine. The aim of our study is tovalorize medicinal and aromatic plants of the Algerian flora. For this reason, we determined the phytochemical composition and antimicrobial activity of the essential oil of Peganum harmala in two different southern regions. Phytochemical screening reveals the presence of flavonoids, alkaloids, saponins, tannins, glycosides, terpenoids and steroids and the absence of anthraquinonesin extracts. The essential oil of P.harmala leaves collected from two different regions (Bougtob and Mechria)was analyzed by GC-MS. The results revealed the presence of 55 and 47 compounds with a yield of 0.217 % and 0.211 %. These essential oils do not contain the same major components for Bougtob region: Carvone 21.15%, Phytol 17.41%, Carbamic acid 3.52%, Lauric acid 2.10%, Thymol 1.77% and Tetrapentacontane 1.32%. For the region of Mechria, Phytol 10.24%, Carvone 5.92%, Tetrapentacontane 2.80%, Phytone 2.18%, Butylated Hydroxytoluene 1.48%, Lauric acid 1.39%. The essential oils which were tested showed variable degrees of antimicrobial activity. In general, Gram-positive bacteria and fungi were found to be more sensitive to the essential oils than Gram-negative bacteria. Keywords Peganum harmala, Leaves, Essential oil and extracts,GC-MS, Phytochemical screening, Antimicrobial activity, southern Algeria. 90 Acta Biologica Slovenica, 2025, 68 (1) Fitokemična sestava in protimikrobna aktivnost eteričnega olja in izvlečkov iz listov Peganum harmala L. v južni Alžiriji. Izvleček Zdravilne rastline imajo široko uporabo v tradicionalni medicini. Namen naše raziskave je valorizirati zdravilne in aromatične rastline alžirske flore, zato smo določili fitokemično sestavo in protimikrobno delovanje eteričnega olja Peganum harmala v dveh različnih južnih regijah. Fitokemijski pregled razkrije prisotnost flavonoidov, alkaloidov, saponinov, taninov, glikozidov, terpenoidov in steroidov ter odsotnost antrakinonov v izvlečkih. Eterično olje listov P.harmala, zbranih iz dveh različnih regij (Bougtob in Mechria), je bilo analizirano z GC-MS. Rezultati so pokazali prisotnost 55 in 47 spojin z izkoristkom 0,217 % in 0,211 %. Ta eterična olja ne vsebujejo istih glavnih sestavin za regijo Bougtob Carvone (21,15 %), fitol (17,41 %), karbamsko kislino (3,52 %), lavrinsko kislino (2,10 %), timol (1,77 %) in tetrapentakontan (1,32 %). Za regijo Mechria, fitol (10,24 %), karvon (5,92 %), tetrapentakontan (2,80 %), fiton (2,18 %), butil hidroksitoluen (1,48 %), lavrinska kislina (1,39 %). Eterična olja, ki so bila testirana, so pokazala različne stopnje protimikrobnega delovanja. Na splošno je bilo ugotovljeno, da so po Gramu pozitivne bakterije in glive bolj občutljive na eterična olja kot po Gramu negativne bakterije. Ključne besede Peganum harmala, listi, eterično olje in izvlečki, GC-MS, fitokemični pregled, protimikrobna aktivnost, južna Alžirija. Introduction Since the highest antiquity, Men have used plants found in nature in several fields, such as perfumery, pharmacology, agri-food, and disease treatments. (Roosta et al., 2017) More than 20,000 aromatic and medicinal plants are used in traditional medicine (Pandey et al., 2013). These plants continue to provide valuable therapeutic agents for both modern and traditional medicine (Bouzouita et al., 2008). Aromatic plants are characterized by the biosynthesis of odorous molecules, which constitute the essential oils known for their antiseptic and therapeutic activity in popu- lar medicine for a long time (Samarth et al., 2017). Essential oils are complex natural substances with well-defined physicochemical characteristics and meet quality criteria that must be known to avoid any risk of toxicity, which may prove dangerous to healthbecausesome essential oils and medicinal plants are very dangerous (Haddouchi et al., 2008) in except to those of some medicinal plants. In addition, these products have a great variability in their chemical constituents, giving them many medicinal and biological properties that should be known and valued (Rashid et al., 2010). The great use of antibiotics in medicine to fight pathogenic micro-organisms is not completely harmless to the body and can cause many undesirable effects (Rudramurthy et al., 2016). Itclearly drives the evolution of resistance (CDCPOIDA, 2013). Epidemiological studies have demonstrated a direct relationship between antibiotic consumption and the emergence and dissemination of resistant bacteria strains.In bacteria, genes can be inher- ited from relatives or can be acquired from nonrelatives on mobile genetic elements such as plasmids. This horizontal gene transfer (HGT) can allow antibiotic resistance to be transferred among different species of bacteria. Resistance can also occur spontaneously through mutation. Antibiotics remove drug-sensitive competitors, leaving resistant bac- teria behind to reproduce as a result of natural selection (Read et al., 2014). Despite warnings regarding overuse, antibiotics are overprescribed worldwide (The Antibiotic Alarm, 2013).Therefore, it is necessary to search for new natural antimicrobial molecules to inhibit various patho- genic microorganisms (Rudramurthy et al., 2016). Algeria, by its geographical location, offers rich and diverse vegetation. A large number of aromatic plants grow there spontaneously. Interest in these plants has continued to grow in recent years. The Algerian flora includes 3300 91 Acta Biologica Slovenica, 2025, 68 (1) species (Quezel& Santa, 1963). Peganum harmala L., com- monly known as Syrian rue or Pègano, is a herbaceous plant belonging to the Zygohpyllaceae family. The primary origin of Peganum harmala is central Asia, but nowadays, it grows in Australia, north of Africa and southwest of Amecira (Bah- mani et al., 2012). It is an herbaceous plant, glabrous, 30 to 60 cm in height with a lignified base, much branched with a winged stem at the top and alternate leaves. It possesses narrow leaves arranged alternately on fleshy, bright green stiff stems (Zargari et al., 1992). The flowers of the plant are isolated in the axils of the leaves,1-2 cm, whitish-green in colour and round seed capsules containing more than 50 seeds. It has a strong, unpleasant odour (Zargari et al., 1992; Marwat et al., 2011). Many related works show that Peganum harmala essential oils have antibacterial, antiviral, antioxidant, anti- parasitic and anticancer activities, among other biological activities. The purpose of our research is toevaluate the chemical composition and antimicrobial activity of essential oils extracted from the leaves of Peganum harmala. This study aims to valorize medicinal and aromatic plants of the Algerian flora in order to find new bioactive natural prod- ucts by the evaluation of the chemical composition and antimicrobial activity of essential oils which are extracted from the leaves of P. harmala collected from two different regions (Bougtob and Mechria). Materials and Methods Plant material The leaves of P.harmala (Figure 1) were collected in April 2020 from the Bougtob region (ElBayed-Algeria) and Mechria region (Naama-Algeria), situated in the South of Algeria. The plant was identified by a botanist from the Faculty of Science and Lifeat the University of Mascara. The leaves were cleaned, shaded, dried, and conserved in a closed and dark room. Essential oil extraction For the extraction of essential oils by hydrodistillation, Cle- venger-type was used (Chouitah et al., 2017), and the dried plant 50g was submitted in a balloon of 500 ml for 3 hours. Sodium sulfate anhydrous(Na2SO4) (used to dry the extract) was added to the essential oil that was recovered and stored in the refrigerator at 4°C and in the dark until use. Figure 1. Peganum harmala L. plant Slika 1. Rastlina Peganum harmala L. 92 Acta Biologica Slovenica, 2025, 68 (1) The yield of essential oil was determined by its contribution to the dry material. R (%) = (m /m0) x 100 Where m: essential oil mass (g), m0: fresh leaves mass (g), R: essential oil yield (%) (Boutekedjiret et al., 2003). Preparation of extracts Ten grams of each powder sample (leaves) were quenched separately in 100 ml of hydromethanolic solution (water/ methanol 20:80 v/v) for 48 hours with stirring to obtain two extracts (Mechria and Bougtob region hydromethanolic extracts). The extracts were then filtered using a Whatman No. 1 filter paper. All filtrates were pressured to a boiling temperature of 50 °C, then they dried at 40 °C and stored at 4 °C until their analysis (Pharmacognosie, 1999). Analysis of the essential oils According to AFNOR NF ISO 280: 1999, essential oils must respond to analytical characteristics that are established by international committees of experts. Standard organoleptic tests and measuring of some physicochemical parameters were performed. Organoleptic characters It is a very important test in determining the quality of an essential oil. It is based on the colour, smell and appear- ance of the essential oil and is carried out according to AFNOR NF ISO 280 (1999). Physical properties of the essential oil Essential oil is said to be miscible with V volume and more ethanol of alkalimetric strength determined at a tempera- ture of 20°C (AFNOR 75-101, 2000).When the mixture of 1 volume of the essential oil with V volumes of this ethanol is clear, and the remainder after gradual addition of ethanol of the same strength up to a total of 20 volumes. Briefly, ethanol of suitable alcoholic strength is gradu- ally added to a test portion of essential oil at a temperature of 20°C, and then their miscibility was evaluated by stirring vigorously during the addition of the solvent. When the obtained solution was perfectly clear, note that the volume (V) of dilution of ethanol was used with continuous addi- tion of solvent in 0.5ml portions up to a total of 20ml, and stirring was performed after each addition.The miscibility of essential oils with ethanol of title t, at a temperature of 20°C, is expressed by One volume of essential oils in V volumes of ethanol with title t. The acid index (NF T 75 103, 1982)expresses the number of milligrams of potassium hydroxide (KOH) required to neutralize the free acids contained in one gram of essential oil. 2 g of essential oil was added to 5 ml of ethanol 95% and five drops of phenolphthalein at 0.2%. The solution was neutralized by the solution of KOH (0.1 mol / l) until a pink colour. We denoted the volume of the KOH solution added. The calculation of AI is given by the formula: AI = 5.61 x V / M;5.61: Corresponds to 0.1 mol / L KOH; M: mass in grams of the essential oil; V: Volume in millilitres of ethanol solution of KOH (0.1 mol / l) used for titration. The ester index value(NFT 75 104, 1982) is the number of milligrams of KOH needed to neutralize the free acids by hydrolysis of esters contained in one gram of essential oil. 2 g of essential oil was added to 25ml of ethanol solution of KOH (0.5 mol / l). It adopted the condenser, placed the ball on the heating mantle and allowed it to heat for one hour. Then, the solution was added to 20 ml of distilled water and five drops of 0.2% PP. The excess KOH solution was titrated with hydrochloric acid 0.5 mol / l. A blank test was carried out under the same conditions and with the same reagents. The calculation of EI is given by the formula: EI = (28.05 x (V0-V1) / M)-IA; 28.05 g / l: corresponding to 0.5 mol / L KOH; M: mass in grams of the test; V0: Volume in ml of the HCl solution (0.5 mol / l) used for the blank; V1: volume in ml of the HCl solution (0.5 mol / l) used to determine the EI of the EO. Physical properties of the essential oil Relative density was performed using a pycnometer with a volume of 1 ml and temperature of 20 °C(NF T 75 111, 1982). Rotator power was obtained according to NF T 75 113 (1982)using a polarimeter-type VISTA C25. Light source (sodium-vapour lamp) for obtaining a light wavelength of 589.3 nm ± 0.3 and a viewing tube 100 ± 0.5 mm in length. The refractive index was carried out using a refract meter to Bellingham type with a precision of ± 0.0002, a direct reading of refractive index between 1.3000 and 1.7000 situated(NF T 75 112, 1977).The freezing point was deter- mined according to AFNOR 75-111 (2000). The essential oil was put in test tubes inside a freezer, accompanied 93 Acta Biologica Slovenica, 2025, 68 (1) by a thermometer, hence the observation of temperature variations accompanying the solidification of the oil, which was cooled slowly and gradually. Phytochemical screening Phytochemical analysis of plant extracts is necessary to reveal the presence of certain chemical families. The detec- tion of different phytochemical components of P.harmala extracts was performed by chemical detection tests based on phenomena of precipitation or colouration using the methods described in the literature.To test for phenols, we added to 2 ml of each extract solution a drop of 2 % FeCl3 alcoholic solution. The positive reaction was expressed by the appearance of a green or blue-black colouration (Adou et al., 2016). For the test of flavonoids, we added 0.5 ml of each extract, ten drops of concentrated hydrochloric acid and a few milligrams of sunflower magnesium. Positive results were indicated by a pink-red or yellow colouration after three minutes of incubation (Haddouchi et al., 2016). To test for tannins, we took 3 ml of extract, placed it in a test tube and diluted with chloroform. 1 ml of acetic anhydride was added. Then, 1 ml of sulphuric acid was poured in care- fully. A green colour was obtained, indicating the presence of tannins (Hossain et al., 2013).For the test for saponins, the dry powder was vigorously mixed with water. A positive result was indicated by the appearance of foam for more than 5 minutes (Joshi et al., 2013).For the test for coumarin glycosides, a few drops of FeCl3 alcoholic solution were added to each extract. The presence of coumarins is indicated by the appearance of a green colouration after the addition of concentrated HNO3 (Joshi et al., 2013).For the test for anthraquinones, approximately 0.5 g of the extract was stirred with 5 ml chloroform for ten minutes and filtered. The filtrate was shaken with 5 ml of ammonia solu- tion. The presence of pink colour in the ammonia phase indicates the presence of anthraquinones. For a combined anthraquinones test, 1 g of powdered extract boiled with 5 ml of 10 % hydrochloric acid for five minutes. The cold filtrate was diluted with chloroform. The chloroform layer was then transferred to a test tube. An equal volume of 10 % ammonia was added to the chloroform extract. The presence of combined anthraquinones was indicated by a pink, red, or purple colour (Abodunrin et al., 2015). For the test for alkaloids, 0.5 g of the extract was mixed with 5 ml of 1 % aqueous hydrochloric acid in a water bath. A few drops of Dragendorff’s reagent were added to 1 ml of the filtrate. Turbidity or precipitation was considered to have a positive result (Ismail et al., 2016).For the test for steroids, 2 ml acetic anhydride and 2 ml H2SO4 were added to 5 ml of the extract. The colour change from violet to blue confirmed the presence of steroids (Kumar, 2015).For the test for proteins, 2 ml of NaOH (4 %) and a few drops of 3 % CuSO4 solution were mixed with 2 ml of solution extract. The presence of protein is indicated by the formation of purple or pink colour (Maria et al., 2018).For the test of quinones, 3 ml of each extract was treated with 3 ml of chloroform, and then the chloroform layer was separated. A volume ofpotassium hydroxide (5 %) was then added to the chloroform layer. The appearance of red colour indicates the presence of quinones (Amezouar et al., 2013). Chromatographic analysis: Gas chromatography/mass spectrometry analysis (GC-MS) The volatile constituents of the essential oil were analyzed by “Shimadzu” GCMS-TQ8030 Gas chromatograph mass spectrometer with a Stabilwax silica capillary column (30 m X 0.32 mm, film thickness, 0.25 µm).Maximum Temp 350°C. The carrier gas was helium, Column Oven Temperature: 50°C, Injection Temperature: 220°C, rate changeof 5°C/ min, Equilibrium Time: 3min, vial capacity: 1.5 ml, and syringe capacity: 10µl. Microbial strains The antimicrobial activity of the essential oil samples was tested against seven different microorganisms: The strains of the international ATCC collection (American Type Cul- ture Collection). Gram+: Staphylococcus aureus ATCC 2592, Bacillus cereus ATCC 11778. Gram- : Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603, Escherichia coli ATCC 25922. Fungus: Candida albicans ATCC 30031, Aspergillus brasil- iensis ATCC 16404. Suspensions were adjusted to 1x107 CFU/ml (equivalent to 0.5 Mc Farland). These strains were maintained on slants of Nutrient Agar for bacteria and Sabouraud Agar for the fungus at 4°C.The tested microbial strains were provided by the Pasteur Institute (Algiers, Algeria). 94 Acta Biologica Slovenica, 2025, 68 (1) Antimicrobial activity The antimicrobial activity of essential oils from P. harmala leaves was evaluated using the paper disk diffusion method (Davis et al., 1994). Petri dishes (90 mm in diam- eter) were prepared with 20 ml of sterile Mueller-Hinton agar and dimethyl sulfoxide (DMSO) solution for bacteria and Sabouraud agar for fungi. Agar plates were stored at 4°C before use (Fauchère et al., 2002). The antimicrobial activity test Saturated with essential oil, the impregnated discs (with 3 µl of essential oil at a final concentration of 1-20µg/ml) were then placed on the surface of the agar (Dean et al., 1987). The bacteria were spread over the discs. , the spreading of the tested product over the entire disc determined the degree of concentration, the micro-organisms grew over the entire surface of the agar except where they encoun- tered a sufficient concentration of the product, which would inhibit their growth after incubation 24h at 37°C (bacteria) and 48h at 25°C (fungi) around of the discs, we observed a clear circular zone formation. This is the zone of inhibition. In addition, this zone of diameter is large; the sensitivity of antibiotics induces more resistance to the bacterium. The diameter of the growth inhibition zone was measured with a calliper and expressed in mm (including the diameter of the 6 mm disc). (Billerbeck et al., 2002; NIST , 2002) The minimum inhibition concentrations (MICs) Were performed according to the Muller-Hinton broth microdilution method in 96 multiwall microtiter plates (Marino et al.,2001).The essential oils were dissolved in aqueous DMSO, and the initial concentration was 25µg/ ml. (Smânia et al., 1995). The initial concentration was serial dilution, which is a two-fold serial dilution. Each well contained 107 CFU/ml of bacteria. After incubation for 24h at 37°C (bacteria) and 48h at 25°C (fungi), the CIM was determined as the lowest concentration at which no visible microorganism growth had occurred. All tests were performed in triplicate (Gilles et al., 2010). Results and Discussion The physicochemical analysis of the leaf extracts of P. har- mala demonstrated the following results: an oily appear- ance, a yellowish colour, and a strong, unpleasant odour. The yield of essential oil obtained by hydro-distillation of dry plants was 0.217 ± 0.03% for the P. harmala Bougtob region and 0.211 ± 0.05% for the Mechria region sample. In a study by Ida et al. in the Bousaada region (South of Algeria), hydrodistillation yielded 0.005% (Ida et al., 2016). The variations found in the yield of essential oil view of our samples compared to some previous studies may be due to some environmental factors (such as soil composi- Figure 3. The yield of essential oil of P.harmala was obtained by hydro-distillation for different regions. Slika 3. Donos eteričnega olja P.harmala, prido- bljenega s hidrodestilacijo za različne regije. 95 Acta Biologica Slovenica, 2025, 68 (1) tion, geographical location and climate conditions), the part of the plant used, the age of the plant and the period of the growing season or even to genetic factors (Hussain et al., 2009; Anwar et al., 2009). Phytochemical screening Phytochemical screening in the leaves of P.harmala showed the presence of flavonoids, alkaloids, saponins, tannins, glycosides, terpenoids and steroids and the absence of anthraquinones for two regions. Phytochemical Compounds of Essential Oil The chromatographic analysis of the essential oil of P.har- mala from the Bougtob region revealed the presence of 55 volatile, constituting 66.59% of the total essential oil recovered from dry material (Table 4, 5). The chemical analysis showed that the characterized essential oil pos- sessed many potentially bioactive substances, which were identified, and the following compounds are representing the major constituents: (1) Carvone 21.15%, (2) Phytol 17.41%, (3) Carbamic acid 3.52%, (4) Lauric acid 2.10%, (5) Thymol Specification Peganum harmala (M) Peganum harmala (B) Density 0.905 0.907 Rotating power +2° + 2° Refractive index +1.4693 + 1.4695 Freezing point -18 -18 Solubility in éthanol 90% 1 :3 1 :3 pH 6.02 6.06 Acid index 0.517 0.521 Ester index 22.7 23.2 Extract constituent Test Observation Extract MM MB Steroids Liebermann–Burchard reaction Purple to blue colour + + Saponins Foam on the surface ++ ++ Tannins Green color +++ ++ Quinones Borntragers test - - Phenols Green-black + + Coumarin glycosides Green to yellow ++ ++ Free anthraquinones Red or pink colour - - Combined anthraquinones Pink, red or purple - - Flavonoids Pink-red or yellow + + Alkaloids Mayer Colored precipitate + + Cardiac glucosides Brown ring ++ ++ Proteins Biuret test Purple or pink colour - - Table 1. Physicochemical composition of Peganum harmala. Tabela 1. Fizikalno-kemijska sestava Peganum harmala. Table 2. Phytochemical screening of Peganum harmala (leaves) hydromethanolic extracts (MM: hydromethanolic extract of Mechria region, MB: hydromethanolic extract of Bougtob region). Tabela 2. Fitokemijski pregled hidrometanolnih izvlečkov Peganum harmala (listi) (MM: hidrometanolni izvleček regije Mechria, MB: hidrometanolni izvleček regije Bougtob). (M): Mechria region, (B): Bougtob region. (-) absence, (+) presence, (+++) presence in high quantity. 96 Acta Biologica Slovenica, 2025, 68 (1) 1.77%, (6) Tetrapentacontane 1.32%, (7) Dihydroactinidiolide 1.21%, (8) 2-Aminobenzaldehyde 1.03%. In contrast, the chromatographic analysis of the essential oil of P.harmala from the Mechria region demonstrated the presence of 47 volatile, constituting 43.6% of the total essential oil recov- ered from dry material. Chemical analysis of essential oil from Mechria region showed that its major products are: (1) Phytol 10.24%, (2) Carvone 5.92%, (3) Tetrapentacontane 2.80%, (4) Phytone 2.18%, (5) Butylated Hydroxytoluene 1.48%, (6) Lauric acid 1.39%, (7) Hexatriacontane 1.27%, (8) 3,5-di-tert-Butyl-4-hydroxybenzaldehyde 1.14% . In a study by Ida et al.(2016), the chromatographic analysis of the essential oil of P.harmala from the Bousaada region demonstrated the presence of 45 volatile constitut- ing 89% of total essential oil showed that its major products are Eugenol 17.5%, Thymol 7%, Carvone 2.7%. The variations found in the chemical composition from the qualitative and quantitative point of view of our samples compared to the study by Ida et al.(2016) may be due to some environmental factors (such as soil composition, geographical location and climate conditions), the part of the plant used, the age of the plant and the period of the grow- ing season or even to genetic factors (Hussain et al., 2009; Anwar at al., 2009). The methods that use water can induce hydrolysis of esters and also rearrangements, isomerizations, racemizations, and oxidations (Bruneton et al., 1993). Khadhri et al.(2011) also say that the variation in the per- centage of the chemical composition of essential oils can be explained by the differences in the geographic origins, climate, nature of the soil, solar radiation, age of the plant and part of plants. All these factors involve the activation or inactivation of certain enzymatic groups, leading to the predominance of a particular biosynthetic pathway (Khadhir et al., 2011). Figure 3. Gas chromatographic profiles the major constituents of Peganum harmala essential oils for two regions; a) Gas chromatographic profiles the major constituents of Peganum harmala essential oils for Bougtob region; b) Gas chromatographic profiles the major constitu- ents of Peganum harmala essential oils for Mechria region. Slika 3. Profili plinske kromatografije glavnih sestavin eteričnih olj Peganum harmala za 2 regiji; a) Profili plinske kromatografije glavnih sestavin eteričnih olj Peganum harmala za regijo Bougtob; b) Profili plinske kromatografije glavnih sestavin eteričnih olj Peganum harmala za regijo Mechria. 97 Acta Biologica Slovenica, 2025, 68 (1) Compound Name Molecular Formula RT (min) Area (%) Eicosane C20H42 13.393 0.30 D - carvone C10H14O 13.758 0.53 Anisole C10H12O 15.986 0.36 5,9-undecided-2-one, 6,10-dimethyl-, (e)- C13H22O 16.655 0.31 Heptadecane, 8-methyl- C18H38 17.742 0.22 Carbamic acid C17H27NO2 17.973 3.52 3,7,11,15-Tetramethyl-2-hexadecen-1-ol C20H40O 18.295 0.24 Caryophyllene oxide C15H24O 18.816 0.13 Ketone C8H12O 19.287 0.18 Ethanone, 1-(1,3a,4,5,6,7-hexahydro-4-hydroxy-3,8-dimethyl-5-azulene)- C14H20O2 19.584 0.56 Hexatriacontane C36H74 19.807 0.25 6,8-nonadien-2-one, 6-methyl-5-(1-methyl ethylidene)- C13H20O 20.196 0.33 2-Benzyl-3-oxo propanoic acid, ethyl ester C13H16O3 20.305 0.20 P-Cymen-7-ol C10H14O 21.670 0.23 Tetrapentacontane C54H110 21.768 0.31 2,6-di(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadiene-1-one C15H24O2 21.847 0.43 Phytone C18H36O 22.205 0.82 Δ-Cadinene C15H24 22.438 0.67 2-aminobenzaldehyde C7H7NO 22.786 1.03 Eugenol C10H12O2 22.944 0.46 Thymol C10H14O 23.514 1.77 Hexacontane C60H122 23.695 0.32 D-(+)-carvone C10H14O 23.978 21.15 Ar-tumerone C15H20O 24.401 0.27 Octaborane B8H12 24.955 0.14 2-Nonen-1-ol, 2-methyl- C10H20O 25.000 0.18 Decanoic acid C10H20O2 25.110 0.32 Pentadecane, 8-hexyl- C21H44 25.495 0.31 Dihydroactinidiolide C11H16O2 25.578 1.21 Quinoline, 6-butyl- C13H15N 25.818 0.19 Phenol, 2,4-bis(1,1-dimethylethyl)- C17H30OSi 25.901 0.36 Dihydroartemisinin, 6-dehydro-5-dehydroxy-3-deoxy- C15H22O4 26.246 0.18 Diethyl phthalate C12H14O4 26.552 0.26 Solasonine C45H73NO16 26.671 0.19 2-propenoic acid, 3-(dimethylamino)-3-ethoxy-, ethyl ester C7H14O3 26.954 0.19 P-vinyl phenol C8H8O 27.174 0.39 Tetracosane C24H50 27.260 0.34 4-quinoline carboxaldehyde C10H7NO 27.315 0.35 Formamide, N-phenyl- C7H7NO 27.695 0.40 Indole C8H7N 27.782 0.32 5.alpha.-cholestan-6.beta.-amine, n,n-dimethyl- C29H53N 27.962 0.31 Methanone, diphenyl- C13H12N 28.154 0.48 N-Hexadecanoic acid C16H32O2 28.385 0.24 Table 3. Phytochemical components identified in Peganum harmala essential oil (Bougtob region) by GC-MS. Tabela 3. Fitokemične sestavine, identificirane v eteričnem olju Peganum harmala (regija Bougtob) z GC-MS. 98 Acta Biologica Slovenica, 2025, 68 (1) Ethyl ether C4H10O 28.501 0.48 Phosphorochloridothioic acid, O,O-dimethyl ester C2H6ClO2PS 28.570 0.19 3,5-di-tert-Butyl-4-hydroxybenzaldehyde C15H22O2 28.615 0.29 Lauric acid C12H24O2 28.791 2.10 Tetrapentacontane C54H110 28.950 1.32 3-methylheptadecane C18H38 29.328 0.30 1,2-Benzenedicarboxylic acid, butyl 8-methylnonyl ester C22H34O4 29.500 0.75 21.xi-methyl-17-isocholest-16-en-3.Beta.-ol C27H46O 29.848 0.35 Triethylene glycol mono dodecyl ether C18H38O4 30.058 0.44 Cyclohexanepropanol, .alpha.,2,2,6-tetramethyl- C13H26O 30.144 0.25 Tetracosane C24H50 30.595 0.33 Phytol C20H40O 30.833 17.41 Compound Name Molecular Formula RT (min) Area (%) Thiirane, octyl- C10H20OS 14.355 0.38 Phosphoric acid C15H41O6PSi4 14.962 0.36 Pentacosane C25H52 15.602 0.33 Methyl 2,6-anhydro-.alpha.-d-altroside C7H12O5 15.853 0.52 Butanedioic acid, decyl phenylmethyl ester C21H32O4 17.162 0.38 2-Methyl-decahydro-4-quinolone C10H9NO 17.268 0.29 Butylated Hydroxytoluene C15H24O 17.960 1.48 β-Ionone C13H20O 18.182 0.38 2-Butenoic acid C18H27NO5 18.288 0.69 2-Nonadecene C19H38 19.147 0.38 Cyclobutaneacetamide, N-(4-methoxyphenyl) C10H13NO2 19.288 0.34 Citronellyl valerate C15H28O2 19.427 0.43 Eicosane C20H42 19.780 0.63 o-Cymene C10H14 20.255 0.24 Hexane, 2,3,4-trimethyl C9H20 20.529 0.23 Phenanthrene, 3-methyl- C15H12 20.785 0.57 Propanoic acid, ethyl ester C5H10O2 21.060 0.21 Pentanoic acid, 5-hydroxy-2-propyl C14H32O3Si2 21.206 0.23 Perhydro-htx-2-one, 2-depentyl-, acetate ester C16H27NO3 21.411 0.23 13-Methyltritriacontane C34H70 21.735 0.25 2,6-Di(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one C15H24O2 21.833 0.62 Phytone C18H36O 22.189 2.18 Carvacrol C10H14O 23.499 0.46 Phytol acetate C22H42O2 23.567 0.37 Tetrapentacontane C54H110 23.657 0.71 Carvone C10H16O 23.960 5.92 Octadecanoic acid C18H36O2 25.090 0.33 Hexatriacontane C36H74 25.472 1.27 2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-, (R)- C11H16O2 25.562 0.37 Quinoline, 6-butyl- C13H15N 25.828 0.32 Table 4. Phytochemical components were identified in the Peganum harmala essential oil Mechria region by GC-MS. Tabela 4. Fitokemične sestavine, identificirane v eteričnem olju Peganum harmala Mechria regija z GC-MS. 99 Acta Biologica Slovenica, 2025, 68 (1) α-Pyrrolidone, 5-[2-butyrylethyl]- C10H17NO2 26.016 0.42 Malonic acid, 6-heptynyl C15H28O4 26.285 0.39 1,2-Benzenedicarboxylic acid, diethyl ester C12H14O4 26.541 0.63 Dotriacontane C32H66 27.233 1.13 Spiro[cyclopropane-1,2'(1'h)-phenanthrene]-1',4'(3'h)-dione, 8'-[(formyloxy) methyl]-4'b,5',6',7',8',8'a,9', C21H28O7 27.281 0.34 1,3,2-Dioxaphosphorinane, 2-(benzyloxy)-2-oxo- C10H13O4P 27.811 0.57 2-(2-Bromo-ethyl)-3-methyl-oxirane C5H9BrO 28.377 0.23 3-Quinuclidinol C7H13NO 28.508 0.47 3,5-di-tert-Butyl-4-hydroxybenzaldehyde C15H22O2 28.600 1.14 Lauric acid C12H24O2 28.773 1.39 Benzenamine, 2,4-dinitro-N-phenyl- C12H9N3O4 28.840 0.36 Tetrapentacontane C54H110 28.924 2.80 Phosphonic acid, bis(1-methylethyl) ester C6H15O3P 29.059 0.40 Pentadecane, 2,6,10-trimethyl- C18H38 29.181 0.55 1,2-Benzenedicarboxylic acid, butyl 8-methylnonyl ester C22H34O4 29.477 0.68 Tetrapentacontane C54H110 30.554 0.76 Phytol C20H40O 30.808 10.24 Antibacterial activity The essential oil of P.harmala leaves of two regions has good inhibitory activity in vitro against the microorganisms tested. However, the microorganisms did not show the same sensitivity to the essential oil. This essential oil has strong antibacterial activity against Gram-positive bacteria, Staphylococcus aureus (inhibition zone 36 mm MIC: 10.5 µg/ml and 33 mm MIC: 11.5 µg/ml) and Bacillus cereus, (inhibition zone 21.7 mm MIC: 15 µg/ml and 18 mm MIC: 17.5 µg/ml) and very strong antifungal, Candida albicans (inhibition zone 25 mm MIC: 09.0 µg/ml and 22.5 mm MIC: 11.5 µg/ml) and Aspargillus brasiliensis (inhibition zone 18 mm MIC: 7.5 µg/ml and 17.5 mm MIC: 10.5 µg/ml). The essential oils have moderate antibacterial activity against Gram-negative bacteria, Pseudomonas aeruginosa (inhibi- tion zone 10.5 mm MIC: 19.5 µg/ml and 8.5 mm MIC: 21 µg/ ml), Klebsiella pneumonia (inhibition zone 13.5 mm MIC: 17.5 µg/ml and 12 mm MIC: 18.5 µg/ml), Escherichia coli (inhibition zone 17 mm MIC: 14.5µg/ml and 15.5 mm MIC: 12 µg/ml) due to the presence of lipopolysaccharide barrier (LPS), which has a role in protecting bacteria against antibacterial agents. Gram-negative bacteria are more resistant to essential oils than Gram-positive bacteria due to the structure of their outer membrane. Thus, the outer membrane of Gram-neg- ative is richer in lipo-polysaccharides and proteins (Toure et al., 2015). Conclusions In this study, we determined the chemical composition and the antimicrobial activity of essential oil from the leaves of Peganum harmala grown in the region of Bougtob and Mechria. Fifty-five compounds havebeen identified in the essential oil of the sample of the first region, Carvone 21.15%, Phytol 17.41%, Carbamic acid 3.52%, Lauric acid 2.10%, and Thymol 1.77%, Tetrapentacontane 1.32% were the major compounds. In the sample of the region of Mechria, 47 compounds were identified when Phytol 10.24%, Carvone 5.92%, Tetrapentacontane 2.80%, Phytone 2.18%, Butyl- ated Hydroxytoluene 1.48%, and Lauric acid 1.39% were the dominant.This difference in composition is due to several factors, such as soil composition, geographical location and climate conditions. Phytochemical screening in the leaves of P.harmala showed the presence of flavonoids, alkaloids, saponins, tannins, glycosides, terpenoids and steroids and the absence of anthraquinones. The antimicrobial effectiveness of the essential oil of P.harmala from southern Algeria has been demonstrated against five bacteria and fungi. This essence showed strong antimicrobial activity against all the tested strains. In general, Gram-positive bacteria and fungi were found to be more sensitive to the essential oils than Gram-negative bacteria. This great power is attributed mainly to their high 100 Acta Biologica Slovenica, 2025, 68 (1) Figure 4. Zones of growth inhibition (mm) are used using a direct contact technique in an agar medium for the antimi- crobial activity of P.harmala essential oils. Slika 4. Območja zaviranja rasti (mm) so s tehniko neposred- nega kontakta v agarskem mediju protimikrobna aktivnost eteričnih olj P.harmala. Figure 5. MIC (µg/ml) using microdilution method in 96 multiwall microliter plates showing antimicrobial activity of P.harmala essential oils. Slika 5. MIC (µg/ml) z metodo mikrorazredčevanja v 96 večstenskih mikrolitrskih ploščah, ki prikazuje proti- mikrobno delovanje eteričniholj P.harmala. 101 Acta Biologica Slovenica, 2025, 68 (1) Figure 6. Comparison of Diameter ofinhibition zone and MIC for the individual bacteria/fungi fortwo regions; a. Comparison of Diameter ofinhibition zone and MIC for the individual bacteria/fungi for Mechria region; b. Comparison of the Diameter of the inhibition zone and MIC for the individual bacteria/fungi for the Bougtob region Slika 6. Primerjava premera inhibicijskega območja in MIC za posamezne bakterije/glive za 2 regiji; a. Primerjava premera inhibicijskega območja in MIC za posamezne bakterije/glive za regijo Mechria; b. Primerjava premera inhibicijskega območja in MIC za posamezne bak- terije/glive za regijo Bougtob 102 Acta Biologica Slovenica, 2025, 68 (1) contents in oxygenated terpenes (Carvone, Phytol and Lauric acid). However, the antimicrobial activity registered confirms some of the traditional uses of the plant in the treatment of some diseases. As a final remark, it is also vital to mention that the essential oil of Peganum harmala is a domain where much remains to be studied to exploit its properties. Author Contributions Conceptualization, G.A. and C.O.; methodology, G.A. and C.O.; software, G.A.; validation, G.A., C.O., K.F. and F.Z.; formal analysis, F.Z.; investigation, C.O. and K.F.; data curation, G.A., C.O., K.F., F.Z., and D.Z.; writing original draft preparation, G.A., C.O., K.F., F.Z., and D.Z.; writing review and editing, G.A., C.O., K.F., F.Z., and D.Z.; visualization, C.O; supervision, C.O; project administration, C.O. All authors have read and agreed to the published version of the manuscript. Acknowledgement Thanks to the members of the laboratories of the Faculty of Natural and Life Sciences at the Universities of Mascara Funding This research received no external funding Data Availability Data are available upon request to the corresponding author. Conflicts of Interest The authors declare that they have no known compet- ing financial interests or personal relationships that could haveappeared to influence the work reported in this paper. 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Acta Biologica Slovenica 68 (1) Received: 11.09.2024 / Accepted: 28.11.2024 / Published: 29.11.2024 https://doi.org/10.14720/abs.68.01.19787 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Soil mesofauna diversity in agricultural systems of Slovenia using the QBS index and its modifications Vid Naglič 1*, Nataša Šibanc 2, Tine Grebenc 2, Irena Bertoncelj 1 Abstract Soil mesofauna plays a key role in maintaining soil health by supporting the decomposition of organic matter, nutrient cycling and the maintenance of soil structure. In this study of Slovenian agricultural ecosystems, we used four modifications of the QBS index, a soil biological quality index based on soil mesofauna. We compared diversity in arable fields under different tillage intensities, a strawberry field and an orchard, managed with either organic or integrated pest management methods (IPM). The results show significant differences in the mesofaunal communities in the soil. Minimum tillage promoted higher biodiversity, especially of Collembola, compared to conventional tillage. In fruit production systems, the ratio of Collembola to Acarina differed from that of arable fields, skewing in favour of Collembola, possibly related to the use of copper-containing pesticides in organic orchards and systemic herbicides in IPM systems. The QBS index values for soil health varied considerably between systems. Only QBS modifications considering the abundances of organisms (QBS- ab and QBS-a) were able to distinguish between different system-management groups. This study provides insights into the limitations of the originally proposed QBS-ar index to discern the effects of farming intensity on the soil mesofaunal community. Results suggest that minimum tillage and organic management practices can promote healthier soil ecosystems, emphasizing the importance of sustainable soil management for the promotion of soil biodiversity. Future research should aim to incorporate a broader range of agricultural practices and assign fauna to a higher taxonomic rank to further explain the effects on soil mesofauna diversity. Keywords Soil health, Soil microarthropods, Biodiversity, Agroecosystems, Tillage intensity, Organic farming 105 Acta Biologica Slovenica, 2025, 68 (1) Introduction The topic of soil health and its indicators has gained con- siderable research interest in the past 20 years, and as soil mesofauna are responsible for several ecosystem services, they have been proposed as a potential soil quality and soil health indicator (Menta & Remelli, 2020). Soil mesofauna plays a key role, being responsible for numerous essential functions such as organic matter decomposition, nutrient cycling, and soil structure maintenance. As such, they are increasingly recognized as valuable indicators of soil quality and health (Menta & Remelli, 2020). The dominance and diversity of specific taxa, notably Acarina (mites) and Collembola (springtails), in agricultural soils have been extensively studied (Behan-Pelletier, 2003). These taxa are not only most abundant but also sensitive to environmental changes, with the potential to serve as reliable bioindica- tors. Previous research (Behan-Pelletier, 2003) has docu- mented different proportions of Acarina and Collembola in various agricultural settings, highlighting their response to different farming practices. For instance, higher abundance of Acarina in organically managed systems compared to integrated pest management (IPM) (Gagnarli et al., 2015) and changes in Collembola abundance as a response to different tillage practices (Vignozzi et al., 2019). Among various indices proposed for assessing soil health, there is the QBS (Qualità Biologica del Suolo) index, which is based on the presence of soil arthropod communities and their level of adaptation to living in soil (Parisi et al., 2005). The QBS index has gained considerable attention in some parts of the world, particularly in Italy, where it originated, and has been applied at the regional level (Albertazzi et al., 2021) to highlight the importance of soil biodiversity for ecosystem services (Menta, Conti, Pinto, et al., 2018). What makes this index advantageous is that it does not require species-level identification, making it a practical tool for large-scale eco- logical assessments and non-taxonomy specialists. The use of indices for describing ecosystems stems from the challenge of grasping the full complexity of these systems, and such indices may offer a simplified Raziskava raznovrstnosti talne mezofavne v kmetijskih ekosistemih Slovenije z uporabo QBS indeksa in njegovih izpeljank Izvleček Talna mezofavna z opravljanjem ekosistemskih storitev razgradnje organske snovi, kroženja hranil in vzdrževanju strukture tal igra ključno vlogo pri ohranjanju zdravja tal. V tej raziskavi slovenskih kmetijskih ekosistemov smo uporabili štiri različice QBS indeksa, ki so bile razvite za namen preučevanja mezofavne v tleh. Raznolikost te skupine živali smo preučevali na njivah z različnimi intenzivnostmi obdelave tal, v nasadu jagod in sadovnjaku, kjer se uporabljajo ekološke ali integrirane metode varstva rastlin (IPM). Rezultati so pokazali statistično značilne razlike v talni mezofavni preučevanih kmetijskih ekosistemov. Pri minimalni obdelavi tal v primerjavi s konvencionalnim oranjem je bila biodiverziteta višja, zlasti pri skupini Collembola. V sistemih pridelave sadja se je razmerje med Collembola in Acarina v prid Collembola razlikovalo od tistega na njivah, kar je verjetno povezano z večjo občutljivostjo Acarina na bakrove pesticide v ekoloških sadovnjakih in sistemske herbicide v IPM sistemih. Vrednosti QBS indeksa so se med sistemi razlikovale. Le QBS različici, ki upoštevata številčnost organizmov (QBS-ab in QBS-a), sta zaznali razlike med različnimi skupinami sistemov in načinov upravljanja. Ta študija kaže na omejitve prvotno predlaganega QBS-ar indeksa za zaznavanje vplivov intenzivnosti kmetovanja na mezofavno v tleh. Rezultati nakazujejo, da lahko minimalna obdelava tal in ekološko upravljanje spodbujata bolj zdrave talne ekosisteme, kar poudarja pomen trajnostnega upravljanja za spodbujanje biodiverzitete v tleh. Da bi lahko natančneje pojasnili vplive kmetijskih praks na raznolikost talne mezofavne, bi morali v prihodnje raziskave vključiti širši spekter kmetijskih ekosistemov ter določiti talne živali do višjih taksonomskih kategorij. Ključne besede Zdravje tal, Talni členonožci, Biodiverziteta, Agroekosistemi, Intenzivnost obdelave tal, Ekološko kmetijstvo 106 Acta Biologica Slovenica, 2025, 68 (1) view aimed at addressing specific research questions. As a result, these indices may not always be effective in detecting significant differences in soil quality between various management practices, especially in ecosystems that are heterogeneous or highly disturbed (Tabaglio et al., 2009). Recent advancements in molecular metabarcoding offer more precise tools for assessing soil biodiversity (Orgiazzi et al., 2015; Guerra et al., 2020). These methods enable more detailed identification, moving from the class- level identification necessary for the QBS index to genus or even species-level identification. Studies confirmed a strong positive correlation between molecular and classi- cal identification tools applied to insects (Jin et al., 2013), and some studies have already been done specifically for soil fauna (Basset et al., 2022). Although genetic studies have provided valuable insights into soil mesofauna, they often miss crucial information about the abundance of these organisms. Additionally, relying solely on DNA analysis may be less effective at detecting recent changes in soil communities, as the persistence of relict DNA from soil organisms can distort our understanding of the current structure of the soil fauna community (Foucher et al., 2020). The QBS index and its variations are employed to assess the impact of agricultural practices on soil biodi- versity and health, a need that has become increasingly important due to the new Soil monitoring law proposed by the EU (General Secretariat of the Council, 2024). In arable fields, the intensity of tillage is a critical factor influencing soil mesofauna. Conventional tillage, which disrupts soil structure, often negatively impacts soil biota, whereas no-till practices tend to enhance soil biodiversity and ecosystem services (Betancur-Corredor et al., 2022). In fruit-growing systems like orchards and strawberry fields, organic farming is generally associated with higher biodiversity due to reduced chemical inputs and a focus on ecological balance. However, the specific contributions of various agricultural practices and soil management approaches influence soil fauna diversity, and consequently, soil health and biodiversity remain unknown. In this study, we aim to explore the diversity and abun- dance of soil mesofauna across three different agricultural ecosystems with two contrasting soil management systems by applying four different variants of the QBS index. The QBS-based approach was selected as an affordable and simple approach. The experiment was set in Slovenia, where highly preserved (extensive) agricultural lands and intensive soil management intermix at small spatial scales. Arable fields, strawberry fields, and orchards were selected as test cultures affecting the soil fauna community, with a focus on varying tillage intensities and the distinction between organic and IPM production methods. Materials and Methods Study sites The experiment was conducted at two locations of the Agricultural Institute of Slovenia’s field research stations: an arable field at the Infrastructure Centre Jablje (46.141204, 14.571509) and a strawberry field and orchard both located at the Infrastructure Centre Brdo (46.166927, 14.680106). The sampling sites within each location were on the same geological base, namely clay gravel and mixed origin gravel soil for Jablje, and clay gravel, sandy loam and clay for Brdo, respectively; same climate conditions Cfbw‘ (mod- erate warm humid climate with warm summers and peak precipitation in one of the autumn months) (Ogrin et al., 2023) according to the Köppen climate classification; with comparable micro-location on flat surface; similar average annual precipitation of 1300-1400 mm and average annual temperatures 10-12°C (ARSO, 2023). The arable field in Jablje was cultivated for five years prior to sampling (since 2018) using three tillage methods: no-tillage without any soil disturbance (0.43 ha), minimal tillage using a disc cultivator or a ripper to a depth of 8-10 cm (0.96 ha), and conventional tillage with ploughing to a depth of 20-25 cm (0.96 ha). The same three-year crop rotation of winter cereal with a cover crop, maize, and soybeans has been practised for all three tillage methods. Fields were fertilized with mineral fertilizers and adapted to crop requirements with average values of 80-100 kg P2O5, 100-130 kg K2O and 160-180 kg N per hectare. Herbicides were applied on average once per year, and fungicides 2-3 times per year in winter wheat crops. All fields were under a large share of crops in the rotation, and no significant pest damage like maize, crimson clover, or soybeans was observed. For the past 50 years, insecticides were used only occasionally, with the frequency once every 4-5 years. The last insecticide application (7.5 g/ha of Lambda-cy- halothrin) was 2 years ago. Lambda-cyhalothrin half-life is around 30-60 days, and considering soil and environ- mental conditions at the study site (warm and microbiolog- ical active soils), insecticide was degraded in a couple of 107 Acta Biologica Slovenica, 2025, 68 (1) months at the latest (Hornsby et al., 1996). The study area was not known to be actively exposed to plastic materials; however, since microplastics and pesticide residues have not been analyzed in this area to date, we cannot rule out their potential presence or influence on mesofauna. Fruit production at the Infrastructure Center Brdo is based on organic and IPM production methods. The straw- berry field was divided into an organic and an IPM section, planted with strawberries (Fragaria x ananassa Duch.; cultivar Clery) two years prior to sampling (in August 2021); four ridges were made, covered with black polyethene and equipped with drip irrigation system. The organic section transitioned through various crops before plant- ing strawberries in 2021, where no fertilizers were used and only plant protection products approved for organic production were used (Pravilnik o ekološki pridelavi in predelavi kmetijskih pridelkov oziroma živil., 2018). In the IPM section, where strawberries were grown according to the recommended crop rotation for eight years before sampling (since 2015), systemic herbicides and only a few fungicides were used before planting. The orchard in Brdo covers 14.9 ha and is also divided into an organic and an IPM section. The Topaz apple variety (Malus domestica var. Topaz) has been cultivated according to the rules of organic production since 2009 (Pravilnik o ekološki pridelavi in predelavi kmetijskih pridelkov oziroma živil., 2018) without the use of herbicides. To remove the weed vegetation under the trees, a rotary tiller is used in combination with a weed brush, which mechanically disturbs the soil to a depth of up to 5 cm. For control of diseases, only solutions approved for organic farming were used, which are mostly copper or sulfur-based (Lešnik et al., 2016; Pravilnik o ekološki pridelavi in predelavi kmeti- jskih pridelkov oziroma živil., 2018). The Gala apple variety (Malus domestica var. Gala) has been grown according to IPM management guidelines with biennial herbicide application according to the Technical guidance (Pravilnik o integrirani pridelavi poljščin, zelenjave, hmelja, sadja in oljk ter grozdja, 2023) in the herbicide strip under the trees for control of the weeds. Figure 1. Location of study sites Jablje and Brdo. Slika 1. Lokacija vzorčnih območij Jablje in Brdo. 108 Acta Biologica Slovenica, 2025, 68 (1) Experimental setup and soil mesofauna sampling Sampling was conducted in three agroecosystems: an arable field, a strawberry field, and an orchard. In the arable field, samples of soil were taken from three tillage methods: no-tillage, minimal tillage, and conventional tillage, with nine samples of each type, giving a total of 27 samples from the arable ecosystem. In the strawberry field, nine samples were taken from each of the organic and IPM sections, giving a total of 18 samples. Two-thirds of the samples were collected from the ridges next to the roots of plants, and one-third of the samples were collected from the interrow spaces between the ridges. In the orchard, we collected nine samples from both the organic and IPM sections, totalling 18 samples, with one-third taken from the root zone of the apple trees and two-thirds from the inter- row areas covered with grass. However, for data analysis, all samples were pooled, resulting in a combined sample size of n=9 for each agricultural system-method group. Standardized soil samples were taken following the QBS sampling protocol (Parisi et al., 2005) using a soil corer with an 11.3 cm diameter to a depth of 10 cm to collect 1 litre of soil in each soil sample. Prior to sampling, the top layer of vegetation was removed using scissors. The soil was collected in plastic bags and transferred to the Kemp- son extractor (ecoTech, Bonn, Germany) (Kempson, 1963), where the soil mesofauna was extracted by air drying the samples for 10 days at 30 °C. Extracted animals were preserved in 70% ethanol. Laboratory analysis of soil mesofauna Extracted animal individuals were identified and counted using stereomicroscope and classified into taxonomic groups (on the level of class or order) and into eco-mor- phologic groups according to the protocol established for the calculation of the QBS index (Parisi et al., 2005). Eco-morphological groups are based on taxonomic groups (class or order level), which are assigned an eco-morpho- logical index (EMI) value between 1 and 20 (possible values are 1, 2, 4, 5, 6, 8, 10, 15, and 20) according to their level of adaptation to the soil environment, with higher values indicating higher adaptation to soil life. The level of adap- tation is determined by morphological characteristics such as body size, pigmentation, length of appendages and presence of eyes. The total number of eco-morphological groups available according to the QBS methodology is 53. Three categories of eco-morphological groups have been proposed by the QBS index authors: epiedaphic (values 1, 2, 4), hemiedaphic (values 5, 6, 8, 10) and euedaphic (values 15, 20) (Parisi et al., 2005). QBS-ar was calculated by summing the highest EMI values of taxonomic groups. For example, Collembola were divided into seven eco-morphological groups with EMI values between 1 and 20. For QBS-ar (Parisi et al., 2005), only the highest EMI value recorded in the sample for Collembola was considered. The second modification was QBS-ar BF (D’Avino et al., 2023), which summarized the EMI values of all present eco-morphological groups in the sample. The third modification, named QBS-a (proposed in this article), considered information on abundance, which was ignored by the previous two, by multiplying the EMI value with the abundance of each eco-morphological group in the sample. The result was divided by 100 for readability. The fourth modification, named QBS-ab, was proposed by Mantoni et al. (2021), where the abundance of each eco-morphological group was logarithmically transformed before being multi- plied by its EMI value. This was done to reduce the influence of the most abundant groups (Acarina and Collembola). Data analysis Cumulative abundance and log-transformed cumulative abundance of taxonomic and eco-morphological groups in all samples were calculated. Species richness rarefaction curves for each agroecosystem were used to determine whether our sampling was thorough with enough collected samples. Principal component analysis (PCA) was used for soil mesofauna community analysis using QBS eco-morpho- logical groups as species. Sample scores on the first and second PCA axes were compared among the seven system-method groups using ANOVA with Tukey post-hoc tests conducted in R software version 4.4.0 ("stats" library). Average species scores (loadings) of epiedaphic, hemie- daphic and euedaphic groups were calculated. In our statistical analysis, we categorized our samples into seven system-method groups, each with nine samples: Arable field-conventional (n=9), Arable field-minimum tillage (n=9), Arable field-no-till (n=9), Strawberry-organic (n=9), Strawberry-IPM (n=9), Orchard-organic (n=9), and Orchard-IPM (n=9). Four modifications of the QBS index were calculated to estimate soil degradation. QBS methodology foresees computation of one QBS value based on EMI 109 Acta Biologica Slovenica, 2025, 68 (1) values of organisms collected in three samples. Therefore, for each system-method group, three QBS index values were obtained out of nine samples. QBS index values as a proxy for soil health were compared between the seven sys- tem-method groups using ANOVA with Tukey post-hoc tests. Results Examination of the cumulative abundance of taxonomic groups showed Acarina were the dominant group in all three arable field management systems, but their dom- inance was less pronounced in orchard and strawberry fields (Figure 2 A). Log transformation of the abundance improved visualization and statistics for the less abundant groups. The more pronounced differences were for Hyme- noptera (ants), which were more abundant in the orchard but not in other systems, and larvae of Coleoptera and Diptera, which were more abundant in arable fields but very few were found in other systems (Figure 2 B). Overall, 63 soil samples from three agroecosystems and 56.6 % (30) soil-adapted eco-morphological groups were recorded from a total of 53 as defined by the QBS index classification of mesofauna. In orchards, rarefaction curves of morpho-taxonomic groups showed sufficient sampling effort, but for arable and strawberry fields, the rarefaction curves did not reach an asymptote (Figure 3). Consider- ing the rarefaction curve slope for the last five samples, we would gain approximately 0.35 and 0.25 additional eco-morphologic groups with each sample in strawberry and arable fields, respectively (Figure 3 A). As QBS method- ology foresees the collection of three samples per sampling site we analysed the richness of eco-morphologic groups in the first three samples of each system-method groups. According to the confidence intervals of these rarefaction curves, the first three samples would detect between 30.5 and 78.0 percent of eco-morphologic groups (Figure 3 B). PCA analysis was used to visualize differences in the community composition of mesofauna (based on eco-morphologic QBS index groups) for all three sampled agricultural ecosystems, with the first axis explaining 15.8 % of the variability and the second axis explaining 14.7 % of the variability (Figure 4). The first PCA axis was mostly determined by the abundance of very numerous Collembola (Figure 4 B) and showed large variability in the community structure of arable fields, especially among minimum tillage samples and lower variability in strawberry field and orchard samples. The seven system-method groups differed signifi- cantly (F=5.3, df=6, p<0.001), with post-hoc tests indicating statistically higher values of PCA scores in arable field under minimum tillage compared to arable conventional tillage and all orchard and strawberry field samples under both organic and IPM management. The average species score for the three groups (epiedaphic, hemiedaphic, euedaphic) had a similar length and direction (Figure 4 A). The second PCA axis separated samples according to less abundant groups such as Isopoda, Diplura, Coleoptera and Symphyla (Figure 4 B). PCA sample scores on the second axis were less variable with no statistical differ- ences between the seven system-method groups (F=1.8, df=6, p=0.106), with one of the orchard samples as a clear outlier (Figure 4 A). In different systems, we observed between 16 (IPM orchard) and 21 (organic orchard) eco-morphologic QBS groups of soil mesofauna (Table 1). The results of the QBS index varied considerably between different modifications of the index. According to both QBS-ar and QBS-ar BF, which do not consider the abun- dance of organisms, the highest values were observed for both the organic strawberry field (Strawberry - eco) and the integrated pest management (IPM) strawberry field (Straw- berry - int), as well as the organic orchard, with the lowest values detected in the arable field and IPM orchard (Figure 5). Differences between the seven system-method groups were only marginally significant for QBS-ar (F=3.2, df=6, p=0.034), with no significant difference detected by post-hoc tests, and were not significant for QBS-ar BF (F=2.1, df=6, p=0.122). On the contrary, the two modifications of the QBS index, which consider the abundance of eco-morphological groups, showed higher values for arable fields compared to fruit production agroecosystems (Figure 5). According to the QBS-ab system method, groups differed significantly (F=8.5, df=6, p<0.001), with the post-hoc test indicating higher values for all arable samples compared to strawberry fields under both organic and IPM management (Figure 5). Post-hoc tests also detected significantly higher values in arable minimum tillage compared to both organic and IPM orchard. The QBS-a also differed significantly between the agroecosystems (F=10.2, df=6, p<0.001), where arable minimum tillage received significantly higher QBS-a values than both fruit production systems under organic and IPM management (Figure 5). Furthermore, the QBS-a index of the arable field under minimum tillage was also significantly higher than the conventionally tilled arable field (Figure 5). 110 Acta Biologica Slovenica, 2025, 68 (1) Figure 2. Cumulative abundance (A) and log-transformed cumulative abundance (B) of 17 taxonomic groups of soil mesofauna in arable field, orchard and strawberry agroecosystems of Slovenia with different management regimes. L indicates larval stages. Slika 2. Kumulativna številčnost (A) in logaritmično transformirana kumulativna številčnost (B) 17 taksonomskih skupin talne mezofavne v njivi, sadovnjaku in nasadu jagod z različnimi režimi upravljanja. L označuje larvalne stadije. 111 Acta Biologica Slovenica, 2025, 68 (1) Figure 3. Rarefaction curves with 95% confidence intervals of morpho-taxonomic groups of soil mesofauna according to QBS methodology in A) three agroecosystems with numbers indicating slope for the last five samples and B) seven system-method groups with numbers indicating the percentage of morpho-taxonomic groups detected in the first three samples per sampling site, as required by the QBS methodology. Slika 3. Rarefakcijske krivulje s 95 % intervali zaupanja za morfo-taksonomske skupine talne mezofavne po metodologiji QBS v A) treh agroekosistemih s številkami, ki prikazujejo naklon za zadnjih pet vzorcev, in B) sedmih sistemsko-metodoloških skupinah s številkami, ki označujejo odstotek morfo-taksonomskih skupin, zaznanih v prvih treh vzorcih na vzorčnem mestu, kot zahteva metodologija QBS. 112 Acta Biologica Slovenica, 2025, 68 (1) Figure 4. Principal component analysis (PCA) scores on the first two axes of A) arable field, strawberry field, and orchard under different management systems (int = IPM; eco = organic; con = conventional tillage; min = minimum tillage; notill = no tillage) with arrows indicating average species scores of epiedaphic, hemiedaphic and euedaphic groups and B) arable, strawberry field and orchard samples with arrows indicating species scores of 5 eco-morphologic groups with the highest species scores on both axes. Slika 4. Rezultati analize glavnih komponent (PCA) na prvih dveh oseh za A) njivo, nasad jagod in sadovnjak pod različnimi režimi upravljanja (int = integrirano varstvo rastlin; eco = ekološko kmetovanje; con = konvencionalna obdelava tal; min = minimalna obdelava tal; notill = brez obdelave tal) s puščicami, ki kažejo povprečne rezultate vrst epiedafičnih, hemiedafičnih in euedafičnih skupin, ter B) vzorce njive, nasada jagod in sadovnjaka s puščicami, ki označujejo rezultate vrst za 5 ekomorfoloških skupin z najvišjimi rezultati vrst na obeh oseh. 113 Acta Biologica Slovenica, 2025, 68 (1) Agricultural ecosystem Management Number of eco-morphological groups Arable field Conventional tillage 17 Arable field Minimum tillage 18 Arable field No-till 19 Strawberry field Organic 19 Strawberry field IPM 20 Orchard Organic 21 Orchard IPM 16 Table 1. Number of detected eco-morphological groups for soil samples collected in three agroecosystems under different management. Tabela 1. Število zaznanih ekomorfoloških skupin za vzorce tal, zbrane v treh agroekosistemih pod različnimi režimi upravljanja. Figure 5. Median and quartile values of four QBS modifications: two without considering animal abundances (QBS-ar, QBS-ar BF), and two considering abundances (QBS-ab and QBS-a) for soil samples from seven system-method groups. The red line indicates a tentative thresh- old value of 93.7 for the QBS-ar index, separating high-quality soils above the threshold from poor-quality soils. Slika 5. Mediana in kvartilne vrednosti štirih različic QBS za talne vzorce iz sedmih sistem-metoda skupin. Dva ne upoštevata abundance živali (QBS-ar in QBS-ar BF), dva pa abundanco upoštevata (QBS-ab in QBS-a). Rdeča črta predstavlja predlagano mejno vrednost QBS-ar (93,7) nad katero so tla visoke kakovosti, pod pa tla nizke kakovosti. 114 Acta Biologica Slovenica, 2025, 68 (1) Discussion Across all studied sites, Acarina and Collembola were the most dominant in samples. These two groups are the two most abundant and diverse taxa of soil mesofauna, which have also been the most investigated (Menta & Remelli, 2020). Their dominance in our samples was unsurprising as it has been reported in similar studies in Europe with the proportion of Acarina between 48-57 % and Collembola around 30 % in vineyards (Gagnarli et al., 2015), with similar proportions in olive orchards (Vignozzi et al., 2019). Similar proportions with dominant Acarina (65 %) and fewer Collem- bola (27.6 %) were reported in forest sites. However, these proportions were inverted in favour of Collembola (56.7 %) compared to Acarina (39.8 %) in cropland and meadows in a systematic study in France (Cluzeau et al., 2012). In our study, the dominance of Acarina over Collembola was much more pronounced in arable fields under all three tillage methods but less pronounced in strawberry fields and orchards. This reduced abundance of Acarina in the strawberry field and orchard could be due to the higher sensitivity of Acarina to soil contaminants such as heavy metals compared to Collembola (Menta et al., 2008), although some experimental studies contradict this (Joimel et al., 2017). Considering the heavy use of copper-based pesticides in fruit production, the lower relative abundance of Acarina compared to Collembola in orchard and strawberry production could indicate their sensitivity to soil contaminants, although further chemical analysis of soil would be needed to confirm this. Addition- ally, the presence of black polyethene mulch in both the organic and IPM sections of the strawberry plantation at the Infrastructure Center Brdo may have contributed to a background level of microplastics in these soils. While no specific analysis of microplastics was conducted in this study, it is worth noting that microplastics are an emerging concern for soil health and may influence mesofaunal communities (Jemec Kokalj et al., 2024). Studies indicate that microplastics can adversely affect soil biodiversity and the health of mesofauna, including groups such as Acarina and Collembola (Shafea et al., 2023). Although assessing microplastic impacts was beyond the scope of this study, future research should consider this factor, particularly in systems where plastic mulching is commonly used. Collembola, on the other hand, responds more to the soil perturbation associated with agricultural practices such as tillage and fertilization (Cluzeau et al., 2012). Reduced tillage had a positive effect on Collembola abundance compared to conventional tillage, with effect varying due to depth, climate, soil texture, but also tillage method and frequency and concurrent herbicide application (Betancur‐ Corredor et al., 2022). The positive effect of reduced tillage on Collembola abundance was also confirmed in our study of arable fields using different tillage methods. PCA analysis showed that tillage intensity had a much greater effect on soil mesofauna community in arable fields than did the type of agroecosystem or the production method (organic or IPM). Variability in the abundance of dif- ferent eco-morphological groups of Collembola seemed to be the most important driver of discrimination between our samples, as indicated by the first PCA axis. This is in line with the results of Chassain et al. (2024), who examined the effects of cropping systems on soil mesofauna density and diversity in 21 fields using practice intensity indicators and indexes and found that the tillage intensity index showed a major impact. In the case of our tested sites, the differences between organic and IPM fruit production varied in more than one management practice, and it is consequently impossible to disentangle their individual effects on soil biota. In the case of the orchard, the two methods differ in the types of pesticides used on trees (synthetic pesticides in IPM and copper-based pesticides in organic), in types of fertilizer (mineral fertilizers in IPM and organic fertilizers in organic) and in the application of herbicides for weed suppression under trees (IPM) and mechanical disturbance for weed removal (organic). In their recent review of below- ground arthropod diversity in conventional and organic vineyards (Di Giovanni et al., 2024), they pointed to unclear management aspects of organic versus conventional farming as the reasons for conflicting responses in soil biota. The same authors also stressed the importance of assessing individual management practices for soil biota functional groups. The QBS-ar index values in our analysis (76–170) were comparable to those reported in other agroecosystem studies (Menta et al., 2018). A tentative threshold value of 93.7 was proposed for QBS-ar to distinguish high-quality soils from poorer ones (Menta et al., 2018). According to this criterion, all sampled management groups except the IPM orchard could be categorised as good-quality soils, with the strawberry field having the highest values. However, the QBS-ar index was not able to distinguish between the different tillage systems due to the high variability, resulting in no statistically significant differences 115 Acta Biologica Slovenica, 2025, 68 (1) between the farming systems. This suggests that while the QBS-ar index is sufficient for distinguishing environments with very different practices, it is not sensitive enough to detect subtle changes resulting from specific management practices such as tillage. Interestingly, the strawberry field — a highly disturbed environment covered with plastic mulch had high QBS-ar values. This could be due to the low number of organisms in the samples, which made it easier for the analyst to identify new ecomorphological groups (EMI) and thus increase the overall score. This highlights a potential limitation of the QBS-ar index: it may overestimate soil quality in disturbed environments where, due to sam- pling artefacts, low organism diversity coincides with an apparently high diversity of EMI. Conversely, environments with a high number of mesofauna — possibly due to high input of organic matter in disturbed environments — do not necessarily reflect better soil health. High numbers of mesofauna feeding on introduced organic matter may be a response to disturbance rather than a sign of a healthy, stable ecosystem. When looking at organism abundance, the QBS results were reversed, with arable areas with different tillage methods showing the highest values. Only the indices that included abundance (QBS-ab and QBS-a) were able to distinguish between different tillage practices, which is consistent with the PCA analysis. These indices, which are sensitive to changes in population density due to agricultural interventions such as tillage, were indeed able to detect abundance-related differences. These results emphasise the complexity of assessing soil health using these indices. Non-abundance indices (QBS-ar) provide information on overall soil quality and ecological stability but may not accurately reflect the nuances of disturbed environments or the effects of organism abundance. Indi- ces with abundance (QBS-ab and QBS-a) provide a more detailed insight into the effects of management actions on soil mesofauna but do not necessarily correlate with improved soil health. Our study emphasises the need for more comprehensive methods to assess soil biodiversity and health. Although QBS indices provide valuable information, they can only capture the dynamics of the soil ecosystem to a limited extent. New techniques such as DNA metabarcoding could provide deeper insights by enabling more accurate identification and quantification of soil organisms and thus improve our understanding of soil health in different agroecosystems. We recommend that researchers carefully consider their objectives and the limitations of each index when selecting a soil biodiversity assessment method. For general assessments of soil qual- ity or ecological stability in very different environments, the QBS-ar may be appropriate but should be used with caution in disturbed environments with low organismal diversity. To detect subtle changes and examine the effects of agricultural practices where organism diversity varies, QBS-ab and QBS-a are more suitable, although they may not fully reflect soil health. Ultimately, a multifaceted approach that combines traditional indices with advanced molecular techniques may be necessary to gain a compre- hensive understanding of soil biodiversity and health. This integrated strategy would contribute to better-informed agricultural management and conservation efforts by enabling a more accurate assessment of the impact of different practices on soil ecosystems. Originally, the QBS methodology foresees the collec- tion of three samples per sampling site, but we increased the effort to nine samples. Rarefaction curves showed that nine samples were sufficient for orchards but not for arable and strawberry fields, where the asymptote was not reached. According to the rarefaction curves, only between 30.5 and 78 percent of eco-morphologic groups would be detected by collecting three samples. This indicates that spatial heterogeneity of soil mesofauna differs between the agroecosystems, and the number of samples should be higher than the three originally proposed in more het- erogeneous ecosystems. Although this study was limited to three agroecosys- tems with only one sampling site, it gives a valuable first insight into the diversity of soil mesofauna within and between agroecosystems. A comparison of different tillage and production methods identified tillage as an important factor determining the soil mesofauna community. How- ever, the complexity of agricultural practices in organic and IPM fruit production makes it impossible to disentangle the environmental factors affecting this community. Supplementary Materials Suppl. Table S1. Abundances of collected taxonomic groups of mesofauna and their proportion of the total sample. Author Contributions Conceptualization, V.N, I.B., N.Š., T.G.; methodology, V.N., I.B., N.Š..; formal analysis, V.N.; writing—original draft prepa- ration, V.N. and I.B.; writing—review and editing, N.Š. and T.G.; visualization, V.N.; All authors have read and agreed to the published version of the manuscript. 116 Acta Biologica Slovenica, 2025, 68 (1) Acknowledgement We would like to thank Agricultural Institute of Slovenia col- leagues Dr. Nika Cvelbar Weber, Dr. Robert Leskovšek and Roman Mavec for establishing and maintaining long term experiments which we sampled. We thank Monika Gričnik for producing the map of the study sites. Funding This study was co-funded by the Slovenian Research Agency (research programs P4-0431 and P4-0107, and research project J4-3098), Slovene Ministry of Agriculture, Forestry and Food (project CRP V4-2221) and Slovenian Research Agency (project CRP V4-2221). 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Acta Biologica Slovenica 68 (1) Received: 18.12.2024 / Accepted: 09.01.2025 / Published: 09.01.2025 https://doi.org/10.14720/abs.68.01.21452 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Review Designer cellulosomes – catalytic nanomachines with significant potential in biotechnology and circular economy Maša Vodovnik 1* Abstract Cellulosomes are multienzyme complexes originally found on the surface of certain anaerobic cellulolytic bacteria and fungi specialized in the degradation of plant cell walls. Recently, the efficiency in lignocellulose conversion and architectural features of these intricate complexes inspired the construction of artificial chimeric complexes for targeted substrate degradation. The simultaneous advancements in synthetic biology, protein engineering and the pursuit of greater sustainability across various industries have highlighted the immense potential of these artificially designed enzymatic complexes for diverse applications. Notably, they hold significant promise for industries specializing in the valorization of plant biomass waste and the production of bio-based renewable energy. The article discusses the main architectural features, design and construction steps, and various biotechnological applications of these intriguing nanomachines. Keywords Designer cellulosomes; lignocellulose valorization; protein engineering; nanobiotechnology, bio-based products 119 Acta Biologica Slovenica, 2025, 68 (1) Sintetični celulosomi – katalitični nanostroji z velikim potencialom v biotehnologiji in krožnem gospodarstvu Izvleček Celulosomi so multiencimski kompleksi, primarno identificirani na površini nekaterih anaerobnih celulolitičnih bakterij in gliv specializiranih za razgradnjo rastlinskih celičnih sten. Zaradi izjemne učinkovitosti pri razgradnji odpornih lignoceluloznih materialov ter njihovih edinstvenih arhitekturnih značilnosti so postali navdih za razvoj sintetičnih hibridnih analogov zasnovanih za ciljno razgradnjo različnih (predvsem odpadnih) lignoceluloznih substratov. Hiter razvoj sintezne biologije, napredki v proteinskem inženirstvu ter vse večja potreba po trajnostnih rešitvah obstoječih izzivov so vodili do spoznanja, da imajo ti umetno zasnovani katalitični nanostroji velik potencial v mnogih industrijskih panogah, ki slonijo na učinkoviti razgradnji lignocellulozne biomase. Še posebno se pričakuje, da bi lahko ti umetni kompleksi pomembno doprinesli v industrijskih aplikacijah, ki temeljijo na valorizaciji odpadne rastlinske biomase, zlasti v sektorjih osredotočenih na pridobivanje obnovljive energije in bele biotehnologije. Članek se osredotoča na glavne arhitekturne značilnosti, zasnovo in ključne korake pri konstrukciji sintetičnih celulosomov ter njihov potencial za različne biotehnološke aplikacije. Ključne besede celulosomi; valorizacija lignoceluloze; proteinski inženiring, sintezna biologija, nanobiotehnologija, bio-osnovani produkti Introduction As the world strives for alternatives to fossil fuels and more sustainable methods for managing agricultural and industrial waste, leveraging the evolutionarily refined natu- ral systems capable of efficiently degrading accumulating plant biomass is becoming increasingly important (Gayathri et al., 2021). Among the most elaborated systems capable of efficient degradation of lignocellulose have been found on the surface of some anaerobic microorganisms. Cellu- losomes are large, multi-enzyme complexes primarily pro- duced by anaerobic cellulolytic bacteria, particularly from the order Clostridiales, inhabiting different environments rich in lignocellulosic materials, such as soil, compost and rumen (Bayer et al., 2004). The pioneering work that estab- lished the cellulosome paradigm began over four decades ago with Bayer and Lamed (Bayer et al., 1983), who first described the extracellular cellulolytic complex of Clos- tridium thermocellum. Subsequent research uncovered similar or even more intricate complexes in other anaer- obic (hemi)cellulolytic bacteria, with the most extensively characterized examples being the cellulosomes of Clos- tridium cellulolyticum, Clostridium cellulovorans, Acetivibrio cellulolyticus, and Ruminococcus flavefaciens and fungi (Artzi et al., 2017). Recently, a minimalistic cellulosome has also been described in the butanologenic bacterium Clos- tridium saccharoperbutylacetonicum (Levi Hevroni et al., 2024). Cellulosomes in these species are specialized for degrading the recalcitrant plant cell wall polysaccharides, including cellulose, hemicellulose, and pectin. Their excep- tional efficiency in breaking down lignocellulosic biomass is due to their highly organized structure and synergistic action of multiple enzymes positioned close to one another (Alves et al., 2020). With the recent rise of white and green biotechnology followed by circular economy movements, these efficient cellulolytic complexes attracted significant attention as potential catalysts for the valorization of lignocellulose waste, not only because of their catalytic efficiency but also due to their specific architectural fea- tures. The modular architecture of structural and catalytic subunits composing these complexes was recognized as potential building blocks for the construction of designer catalytic complexes that can be adapted for specific types of plant biomass (feedstocks), potentially expressed in different hosts and used in various industrial applications (Asemoloye et al., 2023). 120 Acta Biologica Slovenica, 2025, 68 (1) Cellulosome architecture The unique architecture of cellulosomes is critical to their function. The primary components of each cellulosome include one or more structural proteins, scaffoldins, and several catalytic subunits. Primary scaffoldin acts as the backbone of the cellulosome architecture, anchoring the complex to the substrate on the one hand and providing firm docking sites for enzymatic subunits on the other. The enzymatic machinery of cellulosomes comprises a diverse set of carbohydrate-active enzymes (CAZYmes) involved in the degradation of plant cell walls, particularly glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases (Artzi et al., 2017). Furthermore, proteases and their inhibitors (SERPINS) have also been found within some complexes. The spatial organization of the enzymes within the complex minimizes diffusion limitations, enhancing the overall efficiency of the complex (Alves et al., 2021). The main interaction holding together the subunits in cellulosome is the high-affinity interaction between cohesin modules on the scaffoldins and dockerin modules on the enzyme subunits (Type I cohesin-dockerin interac- tion) (as depicted in Fig. 1). This interaction allows dynamic assembly of cellulosome components and adaptation to the enzyme repertoire based on substrate availability. In addition, a different type of cohesin is also present on the secondary, anchoring scaffoldin (Type II), known to bind the main scaffoldin on one side and attach to the bacterial cell wall from the other (Artzi et al., 2017). Some cellulosomes, for example, the complexes identified in R. flavefaciens, are known for even more elaborate structures, involving several scaffoldins binding with each other, resulting in multiplied enzyme-binding sites (Vodovnik et al., 2013; Stern et al., 2016). Furthermore, adaptor scaffoldins have also been discovered that enable switching the substrate specificity of the complex by accommodating different sets of the synthesized enzymes. Most of the scaffoldins (and some enzyme polypep- tides) also contain a carbohydrate-binding module (CBM), which plays a crucial role in targeting and binding to specific carbohydrates, enhancing the catalytic efficiency of the complex by bringing the enzymes in close proximity to the substrate. According to structural properties, CBMs are classified in different families. Some CBM families (for example, CBM 1,2 and 3) target crystalline or amorphous cellulose through hydrophobic stacking interaction and hydrogen bonding with glucose residues. Others, such as CBM families 6 and 22, bind to hemicellulose compo- nents like xylan, mannan or arabinoxylan. Their specificity depends on the sugar composition of the hemicellulose substrate. In addition, pectin-binding CBMs targeting homogalacturonan or rhamnogalacturonan (CBM family 28) have also been identified. Typically, the main scaffoldin identified within the cellulosomal scaffoldins belongs to the family CBM3a, which is known to recognize regular repeating structures on insoluble carbohydrate surfaces, such as crystalline cellulose or chitin. The catalytic (GH) and structural (CBM, dockers, cohesins) modules in cellu- losomes are connected by linker regions, which provide flexibility and enable dynamic movement on the surface of the substrate (Alves et al., 2021). The attachment of cellulosomes to the bacterial cell walls is another critical aspect of their function in biomass degradation. Several strategies are used to anchor scaf- foldins to the bacterial cell wall, including noncovalent interaction via S-layer homology (SLH) domains hydro- phobic anchoring to the lipid bilayer, or sortase-mediated covalent anchoring of the scaffoldins to peptidoglycan (Artzi et al., 2017). Designer cellulosomes Natural cellulosomes, although highly efficient in natural environments, have several constraints that limit their industrial utility, the main three being: (1) non-optimal enzyme composition (the enzyme repertoire of natural cellulosomes is limited to what the host organism nat- urally expresses), (2) environmental constraints (natural cellulosomes are often adapted to specific environmental conditions, such as strict anaerobiosis, which may not align with industrial requirements), (3) lack of flexibility (the fixed enzyme arrangement in natural cellulosomes restricts their adaptability to various substrates) (Lamote et al., 2023). These challenges have been tacked by the advances in molecular and synthetic biology, which paved the way for the construction of artificial complexes with customized enzyme combinations with improved flexibility, efficiency, specificity and stability in industrial conditions (Joseph et al., 2018). Designer cellulosomes (depicted in Fig. 1) are modular complexes with previously designed architecture and composition constructed using synthetic biology and protein engineering tools (Vazana et al., 2013). The construction of such engineered complexes is achievable by leveraging the species-specificity inherent 121 Acta Biologica Slovenica, 2025, 68 (1) in cohesin-dockerin interactions. Apart from the controlled incorporation of target catalytic activities, the engineering of the cellulosomes also allows for the expression and stability optimization of target complexes. The optimization of different subunits can be performed either by rational engineering approach, relying on previous knowledge of the structure-function relationship, or directed evolution, by constructing a library of random mutants followed by rigorous selection based on desired properties of the proteins. Other approaches to increase the stability of designer cellulosomes in industrially-relevant conditions, such as glycosylation, have also been studied (Khan et al., 2020). Several such designer complexes targeted for different biotechnological applications, particularly focused on valorization of (hemi)cellulose biomass, have already been constructed and displayed improved activity, stability, and adaptability across diverse substrates (Wen et al., 2010; Ponsetto et al., 2024). Construction of designer cellulosomes The construction of the designer cellulosomes is a multi- step process that is based on previous knowledge regard- ing the structure-function relationship of different protein components. The main steps in the construction of designer cellulosomes involve (1) selection of target substrate to be degraded (transformed in value-added products); (2) selection of optimal enzyme activities and stochiometry for optimal degradation of the target substrate (based on its composition, i.e. hemicellulose or cellulose prevalence, crystallinity, branching..); (3) complex design: selection of the modules and design of the cellulosome components (scaffoldin design, including the number and specificity of cohesin domains based on the number and type of enzymes to be incorporated and enzymatic subunits design, involving target catalytic domains and compatible dockerin modules); (4) construction of vectors encoding designed modular proteins, molecular cloning to construct Figure 1. Graphic representation of the basic architecture of a designer cellulosome. Doc: dockerin, Coh: cohesin, CBM: carbohydrate-bind- ing module. Differences in colours represent different sources of protein modules within the complex (i.e. the sequence of each module can originate from different species). Slika 1. Grafični prikaz osnovne arhitekture načrtovanega celulosoma. Doc: dokerin, Coh: kohezija, CBM: modul za vezavo ogljikovih hidratov. Razlike v barvah predstavljajo različne vire beljakovinskih modulov v kompleksu (tj. zaporedje vsakega modula lahko izvira iz druge vrste). 122 Acta Biologica Slovenica, 2025, 68 (1) coding sequences of the complex subunits; (5) expression of vector constructs in heterologous hosts (selected based on targeted application) and assembly of the subunits into the final complex, (6) in vitro determination of stability and activity of target designer complexes (Lamote et al., 2023). The selection of protein modules includes three main aspects, starting with scaffoldin and protein modules selec- tion, which is followed by carbohydrate-active enzymes (CAZYmes) and carbohydrate-binding modules (CBMs) selection (based on the target substrate and the desired degree of degradation). In addition, accessory proteins, such as adaptor scaffoldins, may also be added (Stern et al., 2016). Enzymes with different specificities have already been included in such designer complexes. Typically, at least one endo- and exoglucanase pair is needed to target cellulose-rich biomass (Arfi et al., 2014; Vazana et al., 2010), while different types of enzymes targeting hemicellulases (xylanases, arabinases, mannanases), expansins (Chen et al., 2016) or even lignin-targeting laccases may also be added (Fierobe et al., 2001; Fierobe et al., 2005). Further, the selection of cohesin-dockerin pairs and possible linkers is also performed, which does not depend on the substrate composition. The efficiency of designer cellulosomes is influ- enced by many parameters, including the selected docker- ins and linkers, as well as the docking enzyme ratio on the scaffoldin. Recently, the Versatile platform was successfully applied to construct a range of different docking enzymes and scaffoldin variants, facilitating the targeted study of spe- cific parameters influencing the activity of designer cellulo- somes. This platform includes a tile repository composed of dockerins, cohesins, linkers, tags and catalytic modules and allows for the fast and efficient construction of designer cellulosomes, enabling the creation of a practically infinite number of complexes (Vanderstraeten et al., 2022). After selecting the basic components, the final complex architecture is designed by deciding the order of different modules in scaffoldin(s) and designer enzymes, which con- siders enzyme synergy and potential spacial constraints (Caspi et al., 2009). In the next step, modular DNA encoding dockerin-con- taining enzymes and scaffoldin(s) is either synthesized or constructed by different cloning techniques (restriction-di- gestion, CPEC cloning or Versatile shuffling). Different expression systems can be used where each cellulosome subunit lays onto a separate vector (single expression system) or the sequences of multiple components are coexpressed and produced by a single host. Further, different modes of designer cellulosome assembly are possible, such as the “in vitro” assembly by co-incubation of different subunits following their individual expression. The assembled complex can then be charac- terized by native polyacrylamide gel electrophoresis (PAGE) or affinity pull-down. An upgraded version of this process involves the direct assembly on the surface of the expression strain, usually an industrial microorganism, with the ability to directly convert the released sugars to bio-based products, such as ethanol, butanol, etc. (Lamote et al., 2023). So far mostly yeast and solventogenic clostridia have been engineered to display the cellulosomes, particularly Saccharomyces cerevisiae (Tsai et al., 2009; Tsai et al., 2010; Tsai et al., 2013; Fan et al., 2020; Anandharaj et al., 2020, Ma et al., 2024; Sharma et al., 2022), Pichia pastoris (Dong et al., 2020) and Clostridium acetobutylicum (Kovács et al., 2013; Willson et al., 2016), although engineering of some other industrially relevant microorganisms, such as Corynebacterium glutamicum (Lee et al., 2022), has also been reported. Apart from pure solvent-producing cultures, engineered yeast consortium-producing functional mini-cel- lulosomes have also been reported (Goyal et al., 2021). Biotechnological applications of designer cellulosomes The ability to tailor cellulosomes for specific substrates and optimize their efficiency and stability has significant impli- cations for various biotechnological applications based on cellulose waste valorization and the production of bio- based products. One of the most prospective fields that may benefit from the designer cellulosome-based catalysts involves second-generation biofuel production, which relies on converting lignocellulose biomass to solvents or hydrogen. The construction of consolidated bioprocessing systems that simultaneously efficiently degrade various lignocellulose substrates and ferment the released sugars to value-added solvents, like ethanol or butanol, holds significant promise for future sustainable bioprocessing, as it reduces process complexity, minimizes production costs, and enhances overall efficiency (Li et al., 2024). By integrating enzyme production, substrate hydrolysis, and fermentation into a single step, these systems can enable the economically viable conversion of renewable biomass into biofuels and biochemicals, contributing to the development of biorefineries and reducing dependence on fossil fuels (Wen et al., 2020). Furthermore, the trans- 123 Acta Biologica Slovenica, 2025, 68 (1) formation of lignocellulose to other value-added products, including different bio-based chemicals (i.e. lactate, succi- nate), also holds great promise (Liu et al., 2021). In addition, applications in other, more traditional industries, such as textile, paper, feed, and food industries, where (partial) degradation of (hemi)cellulose material enhances the effi- ciency of processes (e.g., polishing of textiles, deinking of paper sludge, or increasing the availability of high-nutrient molecules), may also be improved through the use of these intricate nanomachines. Challenges and Future Perspectives Cellulosomes represent a remarkable natural solution for lignocellulosic biomass degradation, offering a powerful tool for sustainable biotechnology. Their intricate structure, enzymatic synergy, and adaptability to diverse substrates make them ideal for applications ranging from waste man- agement to biofuel production (Xin et al., 2019). However, the construction and industrial application of designer cellulosomes presents several challenges, primarily due to the complexity of designing and assembling these highly specialized enzymatic systems. Key hurdles include the difficulty in optimizing the interactions between the scaffoldin and enzymatic components, ensuring stability and activity under industrial conditions and the need for precise control over the composition and architecture of the complexes. Furthermore, the scalability of designer cel- lulosome-displaying catalysts for large-scale applications, such as in biofuel production or industrial biomass degra- dation, remains a significant obstacle (Wen et al., 2020). By improving enzyme efficiency, tailoring cellulosome designs for specific substrates, and optimizing production systems, these biocatalytic nanomachines can significantly contribute to the development of sustainable utilization of lignocellulosic biomass (Ye et al., 2024). While challenges remain, ongoing advances in genetic engineering, syn- thetic biology, and process optimization promise to unlock their full potential, driving progress in renewable energy and circular bio-economy. Acknowledgement The author thanks the Slovenian Research and Innovation Agency for financial support and N. Lindič for help with graphic design. Conflicts of Interest The author declares no conflict of interest. References Alves, V.D., Fontes, C.M.G.A., Bule, P., 2021. 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Srečanje je sooblikovalo 121 udeležencev - raziskovalci, študenti in drugi strokovnjaki, ki delujejo na različnih področjih znanosti o življenju in znanosti o materialih, ter razstavljavci opreme in aparatur s področja mikroskopije. Predsednica Slovenskega društva za mikroskopijo, dr. Kristina Žagar Soderžnik, je v pozdrav- nem nagovoru izpostavila pomen srečanja za prenos znanja o sodobnih metodologijah na področju mikroskopije, za povezovanje raziskovalcev iz različnih znanstvenih področij ter za izmenjavo izkušenj z uporabniki mikroskopskih analiz v raziskovalno-razvojnih, diagnostičnih, industrijskih ter drugih laboratorijih in ustanovah. Nove metodologije, izzive in rezultate svojega dela so predstavili raziskovalci iz različnih inštitutov, univerz in podjetij iz Slovenije ter nekaj gostov iz tujine, ki intenzivno sodelujejo z našimi laboratoriji. V okviru dveh plenarnih predavanj smo razpravljali o meritvah energijske presnove astrocitov z gensko kodiranimi senzorji na osnovi prenosa energije z resonanco fluorescence (dr. Marko Kreft, Uni- verza v Ljubljani BF in MF, Celica d.o.o.) in o raziskavah perovskitnih feroelektrikov z in situ presevno elektronsko mikroskopijo (dr. Andreja Benčan Golob, Inštitut Jožef Stefan, Ljubljana). V sedmih vabljenih predavanjih smo se seznanili z razvojem in uporabo mikroskopskih analiz v kombinaciji z drugimi metodami na raznolikih področjih: v študijah komunikacije med celicami na osnovi tunelskih membranskih nanocevk, v analizah vpliva podnebnih sprememb na rast dreves, v razvoju novih pristopov za zdravljenje raka z uporabo elektroporacije, v razvoju razno- likih uporab 3D rentgenskega slikanja, v razvoju in kontroli kakovosti v industriji vrhunskih izdelkov iz aluminija, v raziskavah elektrokemijskih reakcij z in situ presevno elek- tronsko mikroskopijo in v naprednih postopkih za karakter- izacijo materialov s katodoluminiscenco. V dveh dneh se je zvrstilo še 56 predstavitev s posterji in 14 krajših predavanj o novostih na področju priprave in mikroskopskih analiz vzorcev, kar je spodbudilo zelo živahne razprave o novih metodoloških možnostih, ki so na razpolago v labora- torijih v Sloveniji, o najnovejšem razvoju metodologije v globalnih raziskavah in o spektru možnih uporab novih tehnik. V znanostih o življenju je zelo aktualno področje razvoja različnih pristopov za integracijo in korelacijo več načinov slikanja, ki so komplementarni z vidika različnih vrst pridobljenih informacij, različnih ločljivosti in velikosti analiziranega vzorca. Inovativni postopki integrativne mikroskopije bioloških objektov nam omogočajo celovit vpogled v biološke strukture in procese. Poleg tega je za pridobivanje kvantitativnih rezultatov iz slikovnih podatkov ključnega pomena napredna analiza slik. Srečanje smo zaključili z razpravo o pomembnem dogodku, ki ga Slov- ensko društvo za mikroskopijo organizira v naslednjem letu, to je mednarodni kongres 17th Multinational Congress on Microscopy (https://17mcm.si/). Nada Žnidaršič, Rok Kostanjšek, Polona Mrak Oddelek za biologijo, Biotehniška fakulteta, Univerza v Ljubljani Acta Biologica Slovenica 2025 Vol. 68 | Št. 1