Investigation of antimicrobial activities and molecular characterization of the species belong to Origanum, Thymus and Thymbra genera by ISSR

Molecular Biology Reports (2023) 50:289–298 https://doi.org/10.1007/s11033-022-07923-y ORIGINAL ARTICLE Investigation of antimicrobial activities and molecular characterization of the species belong to Origanum, Thymus and Thymbra genera by ISSR Sibel Kerem1 · Nezahat Koşar4 · Fetullah Tekin2,3 · Ayşe Semra Güreser4 · Özlem Özbek1 Received: 19 February 2022 / Accepted: 6 September 2022 / Published online: 4 November 2022 © The Author(s), under exclusive licence to Springer Nature B.V. 2022 Abstract Background: The aim of this study is to investigate the antimicrobial activities of the species belonging to the genera Origanum L., Thymus L., and Thymbra L. in the Lamiaceae family and molecular characterization using ISSR markers and to determine the correlations between anti-microbial activities of the plant extracts and ISSR loci. Methods and Results Anti-microbial active extracts were obtained after 24-hours extraction using either of the three different solvents (ethanol, hexane, and chloroform) from the plants using the Soxhlet device. The effects of extracts on the bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis) were determined using the disc-diffusion method. The species Thymbra spicata var. spicata L., Thymus vulgaris L., Thymus citriodorus, Thymus cilicicus, Origanum syriacum L., and Origanum vulgare L. subsp. hirtum displayed significant anti-microbial activities, while the Origanum minutiflorum, Origanum onites L., Origanum saccatum and Origanum vulgare L. ssp. gracile displayed less activities on the bacterial strains. The plant species under study had a high level of genetic diversity. Significant correlations were determined between the anti-microbial activities of the plant species and the ISSR loci. Conclusion Staphylococcus aureus was the most sensitive and Pseudomonas aeruginosa was the least sensitive strain. The ethanol and chloroform extracts were the most effective solvents. ISSR markers were successful for determining high levels of genetic diversity and clustering the species belonging to the genera Origanum, Thymus, and Thymbra. Conducting molecular marker analyses facilitated in distinguishing the species correctly for molecular breeding studies. The studies identified the antimicrobial activities of the plants against the bacteria used in the study and suggested their potential role in the pharmaceutical industry. Keywords Medicinal and aromatic plants · Lamiaceae · Secondary metabolites · Biomolecules · Alternative Medicine · Breeding · Genetic diversity · Disc-diffusion method Introduction Özlem Özbek ozbekozlem@gmail.com 1 Faculty of Science and Arts, Department of Biology, Hitit University, Çorum, Turkey 2 Faculty of Science and Arts, Department of Molecular Biology and Genetics, Hitit University, Çorum, Turkey 3 Republic of Turkey, Ministry of Agriculture and Forestry GAP International Agricultural Research and Training Center, Diyarbakır, Turkey 4 Faculty of Medicine, Department of Microbiology, Hitit University, Çorum, Turkey The Lamiaceae (Labiatae) family includes many medicinal and aromatic plants. The Lamiaceae—Mint family (lamium, gullet, after the shape of the corolla tube or old Latin name used by Pliny) contains 236–238 genera/7.587 species in worldwide [1], and 44 taxa with 33 species, 22 of which are endemic in Turkey [2]. The genera Origanum L., Thymbra L. and Thymus L. are included among the prominent medicinal and aromatic plant species within the Lamiaceae family. The genus Origanum L. belongs to tribe Mentheae including 42 species and 18 hybrids widely distributed in Eurasia, Mediterranean, Euro-Siberian, Irano-Siberian regions, and North Africa [3]. According to Ietswaart [3] classification, 13 290 the genus Origanum L. subdivided into 10 sections. The genus Thymbra L. is represented by two species; Thymbra spicata L. which has two varieties; var. spicata L., and var. intricata, and Thymbra sintenisii, which has two subspecies ssp. sintenisii and ssp. isaurica in Turkey [4]. T. spicata L. is naturally cultivated in Southeastern Anatolia, coastal areas of Thrace, Aegean, and Mediterranean region of Turkey [2]. The genus Thymus L. consists of over 400 species. It includes herbaceous annuals and perennial plants that are widely used, for medicinal and non-medicinal purposes. These plants are widely distributed throughout the Old World [5]. The scientific researches conducted in the last few decades, has been reported that medicinal and aromatic plants contain secondary metabolites with antimicrobial activities and that there are biomolecules among them that have significant effects, in the development of alternative treatment methods [6, 7]. The essential oils extracted from the medicinal and aromatic plants had antimicrobial activities on different microorganisms (Bacteria, Protista and Fungi) revealed in many previous studies. The correlation between the data of chemical contents (thymol, carvacrol, linalool, geraniol, α-terpineol, thuyanol-4, geraniol, and p-cymene) and molecular characterization of genotypes using RAPD, and ISSR has been performed for several aromatic and medicinal plants including T. vulgaris, Thymus (T. daenensis Cĕlak. (two populations), T. fallax Fisch. & C. A. Mey., T. fedtschenkoi Ronniger, T. migricus Klokov & Des.Shost., and T. vulgaris L. previously by several researchers [8–10]. ISSR is a DNA based marker located between microsatellite loci and the long SSR-based primers of di-, tri-, tetra-or penta-nucleotide motifs that were designed as ISSR primers [11]. ISSRs have the advantages of simplicity, acceptable stability, and high reproducibility [12, 13] due to high primer annealing temperature [14, 15], highly polymorphic, cost effective, requiring no prior information of the sequence [16]. DNA markers have been used successfully in genetic variation studies, gene mapping, germplasm identification, and fingerprinting construction as well ISSR [12, 13]. The genetic diversity was investigated in the species belonging to the genera Origanum L., Thymus L., and Thymbra L. by using different molecular markers in previous studies. Amplified fragment length polymorphism (AFLP) markers were used in the genus Origanum L. and Thymus L. including their available species in Egypt [17], ISSRs for the assessment of genetic diversity and relationships in Thymus L. species [18], microsatellites in Thymus cilicicus Boiss. & Bal. [19]. SRAP and EST-SSR molecular markers used for the specimens from the Mediterranean, Eastern Anatolian, Central Anatolian, and Black Sea regions of Turkey [20], simple sequence repeat (SSR), and cleaved amplified polymorphic sequence (CAPS) markers for Origanum 13 Molecular Biology Reports (2023) 50:289–298 L. species from Antalya in Turkey [21]. SSRs and randomly amplified polymorphic DNA (RAPD) markers were used for revealing phylogenetic relationships within Origanum vulgare ssp. hirtum populations and among the Origanum L. species [22], while intraspecific diversity and relationship between subspecies of Origanum vulgare was analyzed by using AFLP and SAMPL markers [23]. The genetic relationships among 12 Thymus L. taxa from Portugal were investigated by AFLP [24], whereas El Sherbeny et al. [25] assessed the genetic diversity and relationship among Thymus L. sp. using RAPD and the inter-simple sequence repeats (ISSR) markers. In this study, we aimed to investigate antimicrobial activities of ten different species belong to the genera Origanum L., Thymus L. and Thymbra L. on four different bacterial strains, and molecular characterization of the species by ISSR markers and their correlation with the antimicrobial activities of the species. Materials and methods Materials Plant samples In this study, ten different species belonging to three different genera; Origanum L., Thymus L. and Thymbra L.were used for the antimicrobial analyses. The plant samples were collected from different locations and propagated at the trial fiels of the Directorate of GAP International Agricultural Research and Training Center of the Turkish Republic Ministry of Agriculture and Forestry. Detailed information about the plant samples used in the study is given in Table 1. Methods Soxhlet extraction The air- dried aerial parts (leaves and stems) of harvested whole plant materials (30g) were ground using liquid nitrogen (-196°C) in a ceramic mortar. The powder of grinded leaves was used for the extraction throughout 24h by the Soxhlet device. In the study, three different solvents; chloroform (≥ 99.5%), ethanol aqueous solution (96%), and hexane were used for extraction. When the extraction was completed, the solvent containing the extracted solutes was allowed to evaporate until 30 mL left in the flask of the Soxhlet device. Then, the solution containing the extract was transferred to the fresh tubes (50 mL), which Molecular Biology Reports (2023) 50:289–298 Table 1 The species used as plant materials and their original collection locations Species name Location Mardin Darülzaferan Thymbra spicata var. spicata L. Yalova Atatürk Bahçe Thymus vulgaris L. Kültürleri Merkez Araştırma Enstitüsü Yalova Atatürk Bahçe Thymus citriodorus (Schreb) Kültürleri Merkez Araştırma Enstitüsü Kahramanmaraş Thymus cilicicus Boiss. & Bal. Adana Origanum syriacum L. Origanum minutiflorum O. Schwarz & P.H. Isparta Sütçüler Davis İzmir Origanum onites L. Antalya Origanum saccatum P.H. Davis Origanum vulgare L. ssp. gracile (C. Koch) Şırnak Ietswaart Tekirdağ Origanum vulgare L. ssp. hirtum (Link) letswaart were centrifuged 1min. at 2000rpm. The supernatant was removed to the fresh amber color glass bottles, allowed to evaporate until the solid extracts left, and kept at + 4°C until it was used. For antimicrobial analyses, the solute extracts were resolved with the same solvent (5 mL) used for extraction, after that solutions were filtered using micro filters with 0.22μm pore sizes into the sterilized fresh amber color glass bottles with caps, and kept at + 4°C until used. Bacterial strains and growth medium Four bacterial strains, two Gram (+) (Enterococcus faecalis ATCC 29,212 and Staphylococcus aureus ATCC 29,213) and two Gram (-) (Pseudomonas aeruginosa ATCC 2753 and Escherichia coli ATCC 25,922) were used for the antimicrobial analyses. Bacterial strains were cultivated in different growing media in the following order; blood agar, Luria-Bertani (LB), nutrient broth, and nutrient agar at their respective optimal growth temperatures (37°C). KirbyBauer Disk Diffusion Susceptibility Test Protocol was used as described by Hudzicki [26] for antimicrobial analyses. The bacterial suspensions were adjusted as 0.5 McFarland standard, which is equivalent to a bacterial suspension containing between 1 × 108 and 2 × 108 CFU/mL of E. coli by using sodium chloride physiological solution. DNA isolation The genomic DNA was isolated as described by Kidwell and Osborn [27] from the dried aerial parts (leaves and stems) of the plants belonging to ten different species understudied. 291 Amplification of ISSR loci For the PCR reactions; 1x Taq buffer (10×), 3 mM MgCl2 (25 mM), 200 µM dNTPs (10 mM each), 0.2 u of Taq DNA polymerase (5u/µL, Thermo), 0.2 µM ISSR primer (10 pMol, Query, Alpha DNA), 1 µL template DNA (10–40 ng) were mixed in the final concentration and distilled water was added up to 20 µL. A thermocycler PCR system (Thermo Scientific) was used to carry out the PCR amplifications. Ten ISSR primers were used for the molecular characterization of the genotypes (Table S1). The thermal program for the DNA amplification was managed as one cycle for 4 min at 94°C, 35 cycles for denaturing in 45 s at 94°C, for annealing in 30 s at 58°C, and for extension in 2 min at 72°C, followed by one cycle for final extension in 7 min at 72°C. The PCR amplicons were run along with 100 bp DNA molecular size marker (GeneDireX) on 1.3% agarose gel (Sigma), and electrophoresis carried out at 60 mA / 120 V for 2-2.5 h. The ethidium bromide (10 mg/mL) staining was used to visualize the amplified fragments, and the pictures were taken under UV light (DNR bio-imaging system). Statistical analyses One-way ANOVA For the statistical analyses, the means of the data were used. Inter-group comparisons were assessed using one way ANOVA followed by Tukeys’s HSD (Honest Significant Difference) multiple comparison post-hoc test. Genetic diversity The PCR amplification of ISSR fragments were scored as binary data; 1 for present and 0 for the absent fragment and the binary data were computed using the software PopGene (ver. 1.32) [28]. For the genetic diversity estimates, the mean number of alleles per locus (na), the mean number of effective alleles per locus (nea), and the mean values of the genetic diversity (h) were calculated for all the species [29]. For dominantly inherited DNA markers, the genetic differentiation between the groups is usually estimated with GST [29], which displays how the genetic variation is partitioned within and between groups. GST value was used to estimate the gene flow (Nm) between the groups. The phylogenetic relationships between the species were displayed by a dendrogram constructed according to the genetic distances [29]: Method = UPGMA (unweighted pair group method with arithmetic mean) based on the ISSR data, adopted from the NEIGHBOR procedure of the PHYLIP Version 3.5. 13 292 Results Antimicrobial activity According to the antimicrobial activity analyses, the ethanol and chloroform extracts of Thymus vulgaris L. and Thymbra spicata ssp. spicata L., and the ethanol extracts of Thymus citriodorus had high antimicrobial activities on Gr(-) bacterial strains and Gr(+) bacterial strains (Table S2). The hexane extracts of Origanum vulgare L. ssp. hirtum, Origanum onites L. and Origanum vulgare ssp. gracile species displayed most effective results on Gr(-) bacterial strains and Gr(+) bacterial strains. The chloroform extracts of the species Origanum minutiflorum, Origanum vulgare ssp. gracile, Origanum saccatum, Origanum syriacum L., Origanum onites L. and Thymus citriodorus showed higher activities on Gr(-) bacterial strains and Gr(+) bacterial strains. According to antimicrobial activity results, when 15 µL, 20 µL, and 25 µL ethanol extracts were applied on the bacterial strains, the highest effective antimicrobial activities were observed on S. aureus and the lowest antimicrobial activities were observed on E. coli and P. aeruginosa in general (Table S2). When 15 µL hexane extracts were applied on the bacterial strains, the highest effective antimicrobial activity were observed on S. aureus, and there were no antimicrobial activities on E. faecalis, E. coli, and P. aeruginosa except a few ignorable values. However, when 20 µL and 25 µL hekzan extracts were applied on the bacterial strains, the effects of antimicrobial activities increased in all the bacterial strains, but the increases were not significant on the bacterial strains other than on S. aureus (Table S2). When 15 µL chloroform extracts were applied on the bacterial strains, the effects of antimicrobial activities were ranked from high to low for S. aureus, E. faecalis and E. coli, respectively, but except a few ignorable values, there was no antimicrobial activity on P. aeruginosa. When 20 µL and 25 µL chloroform extracts were applied on the bacterial strains, the effects of antimicrobial activities increased in all the bacterial strains (Table S2). One-way ANOVA and the post hoc test-Tukey’s HSD. Variation according to four different bacterial strains Two different degrees of freedom (df) values are required to find the critical value, dfbetween groups (BG) and dfwithin groups (WG). F (df(BG),df(WG)) = F (3, 32) = 2.901 (critical value in t table) (Table 2). According to ANOVA, the F values calculated for the antimicrobial activities of ten species on four bacterial strains were greater than the critical value > 2.901, thus the null hypothesis was rejected (H0 = µE. faecalis = µS. 13 Molecular Biology Reports (2023) 50:289–298 = µE. coli = µP. aeruginosa) and the alternative hypothesis was accepted (H1 = not all µs were equal) (Table 2). The effects of antimicrobial activities of all the species on the four different bacterial strains were not the same, but different from each other. These differences were due to the fact that the antimicrobial activities of the species [Thymbra spicata var. spicata L., Thymus vulgaris L., Thymus citriodorus, Origanum syriacum L. and Origanum saccatum) on the bacterial strain S. aureus were different from the effects on the other bacterial strains [E. faecalis, E. coli, and P. aeruginosa] (Data not given). The effects of antimicrobial activities of Thymus cilicicus and Origanum minutiflorum on the bacterial strain S. aureus were different from that on E. faecalis and P. aeruginosa. The effects of antimicrobial activity of Origanum onites L. on the bacterial strains S. aureus, E. faecalis and E. coli were different. The effects of antimicrobial activities of the Origanum vulgare L. ssp. gracile on the bacterial strains S. aureus, E. faecalis, and E. coli were different, while the effects on S. aureus and P. aeruginosa bacterial strains were different, there were no differences on the E. faecalis, E. coli, P. aeruginosa bacterial strains. Finally, The effects of antimicrobial activities of the Origanum vulgare L. ssp. hirtum on the bacterial strains S. aureus, E. faecalis, E. coli, and P. aeruginosa were different, while there were no differences between the effects on the E. faecalis and P. aeruginosa. aureus Variation according to three different solvents The antimicrobial activities of the extracts obtained with three different solvents indicated that the critical value was 3.285 for F (2, 33) according to one-way ANOVA (Table 3). The F values calculated for the effects of antimicrobial activities of the extracts with three different solvents on the four bacterial strains were greater than the critical value > 3.285, thus the null hypothesis was rejected (H0 = µethanol = µhekzan = µchloroform) and the alternative hypothesis was accepted (H1 = not all µs were equal). The effects of antimicrobial extracts of all the species [Thymbra spicata var. spicata L., Thymus vulgaris L., Thymus citriodorus, Origanum syriacum L., Origanum minutiflorum, and Origanum saccatum] obtained with three different solvents on four different bacterial strains were different from each other (Table 3). The effects of antimicrobial activities of ethanol and chloroform extracts of the species [Thymus vulgaris L., Origanum syriacum L., and Origanum minutiflorum] on four different bacterial strains were different from each other. The effects of antimicrobial activities of the extracts of the species [Thymus citriodorus, and Origanum saccatum] obtained with ethanol and hexane on the four different bacterial strains were different from each other. Molecular Biology Reports (2023) 50:289–298 293 Table 2 The significance of the effects of antimicrobial activities of the plant extracts obtained with three different solvents on four bacterial strains by one-way ANOVA (Sum of Squares SS, Degree of Freedom df, Mean Square MS, F: Critical value, p: Sigma Between Groups BG, Within Groups WG) Plant Species SS df MS F Sig. 1989.50 3 663.17 11.21 0.00 Thymbra spicata var. spicata BG L. WG 1892.58 32 59.14 Total 3882.08 35 BG 5113.46 3 1704.49 11.43 0.00 Thymus vulgaris L. WG 4773.14 32 149.16 Total 9886.61 35 4091.73 3 1363.91 14.46 0.00 Thymus citriodorus (Schreb) BG WG 3018.09 32 94.32 Total 7109.82 35 BG 443.24 3 147.75 5.03 0.01 Thymus cilicicus Boiss. & Bal. WG 939.46 32 29.36 Total 1382.70 35 BG 935.22 3 311.74 7.46 0.00 Origanum syriacum L. WG 1337.10 32 41.78 Total 2272.32 35 BG 1151.43 3 383.81 9.16 0.00 Origanum minutiflorum O. Schwarz & P.H. Davis WG 1341.21 32 41.91 Total 2492.64 35 BG 3054.48 3 1018.16 41.49 0.00 Origanum onites L. WG 785.25 32 24.54 Total 3839.74 35 BG 1747.57 3 582.52 17.11 0.00 Origanum saccatum P.H. Davis WG 1089.15 32 34.04 Total 2836.72 35 BG 1697.27 3 565.76 30.38 0.00 Origanum vulgare L. ssp. gracile (C. Koch) Ietswaart WG 595.93 32 18.62 Total 2293.20 35 BG 1975.72 3 658.57 50.03 0.00 Origanum vulgare L. ssp. hirtum (Link) letswaart WG 421.27 32 13.16 Total 2396.99 35 *p < 0.05 (at significant level) Variation according to application of extracts with three different volumes The critical value was found as 2.305 for F (8, 27) according to one-way ANOVA. The F values calculated for the three different volumes of the extracts applied on the four different bacterial strains were smaller than the critical value < 2.305, thus the null hypothesis was accepted (H0 = µ15µl = µ20µl = µ25µl), and the alternative hypothesis was rejected (H1 = not all µs were equal) (Table S3). Pearson’s (rP) correlations. Pearson’s correlations were performed between the responses of the bacterial strains to antimicrobial activities of the extracts and ISSR loci obtained from ten species. When the Pearson’s correlation results were evaluated, the significant positive correlation values were observed between some ISSR loci and the responses of the bacterial strains to the extracts obtained by different solvents and a few examples were given here. The significant positive and negative correlation values between ISSR loci and antimicrobial activities were observed as; for the loci 817 − 12, 826 − 18 and 865-4 rp = 0.936 (at p < 0.00 significant level) in P. aeruginosa, and the loci 808-4 and 808 − 11 rp = -0.774 (at p < 0.01 significant level) in E. coli for 20 µL ethanol extract (Table S4); for the loci 808-5 rp = 0.999 (at p < 0.00 significant level) in P. aeruginosa, and 808 − 20 rp = -0.834 (at p < 0.00 significant level) in E. coli for 15 µL chloroform extract (Table S5); for the loci 810-6 rp = 0.872 (at p < 0.00 significant level) in S. aureus, and 808 − 20 rp = -0.647 (at p < 0.04 significant level) in E. coli, for 15 µL hexane extracts (Table S6). Molecular analyses Ten ISSR primers produced 170 loci, of which 161 (94.71%) were polymorphic and 9 (5.29%) were monomorphic in the PCR analyses. The mean number of alleles, effective alleles, and genetic diversity values were observed as na = 1.95, nea 13 294 Molecular Biology Reports (2023) 50:289–298 Table 3 The significance of the antimicrobial activities of the plant extracts according to the solvent types (ethanol, chloroform, and hekzane) by one-way ANOVA (Sum of Squares SS, Degree of Freedom df, Mean Square MS, F: Critical value, p: Sigma Between Groups BG, Within Groups WG) SS df MS F Sig. 1303.21 2 651.60 8.34 0.00 Thymbra spicata var. spicata BG L. WG 2578.87 33 78.15 Total 3882.08 35 BG 2696.15 2 1348.07 6.19 0.01 Thymus vulgaris L. WG 7190.46 33 217.89 Total 9886.61 35 1610.17 2 805.09 4.83 0.01 Thymus citriodorus (Schreb) BG WG 5499.65 33 166.66 Total 7109.82 35 186.70 2 93.35 2.58 0.09 Thymus cilicicus Boiss. & Bal. BG WG 1196.00 33 36.24 Total 1382.70 35 BG 833.32 2 416.66 9.56 0.00 Origanum syriacum L. WG 1439.00 33 43.61 Total 2272.32 35 BG 747.30 2 373.65 7.06 0.00 Origanum minutiflorum O. Schwarz & P.H. Davis WG 1745.34 33 52.89 Total 2492.64 35 BG 400.45 2 200.23 1.92 0.16 Origanum onites L. WG 3439.28 33 104.22 Total 3839.74 35 BG 500.75 2 250.38 3.54 0.04 Origanum saccatum P.H. Davis WG 2335.97 33 70.79 Total 2836.72 35 BG 286.23 2 143.12 2.35 0.11 Origanum vulgare L. ssp. gracile (C. Koch) Ietswaart WG 2006.97 33 60.82 Total 2293.20 35 62.96 2 31.48 0.45 0.64 Origanum vulgare L. ssp. hir- BG tum (Link) letswaart WG 2334.03 33 70.73 Total 2396.99 35 *p < 0.05 (at significant level) = 1.47 and h = 0.30 respectively (data not given). Figure 1. displays the band patterns produced in ten species understudy by using the primer UBC-808. The highest genetic distance value (D = 0.63) was observed between the species Thymus cilicicus and Origanum onites, while the lowest genetic distance value (D = 0.16) was observed between Origanum saccatum and Origanum vulgare L. ssp. hirtum (data not given) (Fig. 2). The dendrogram, constructed based on the ISSR data, displayed that the cultivated species Origanum onites L. was clustered separately from the wild species. In the wild species, Thymus cilicicus was the distant species to the other wild species. Thymbra spicata var. spicata L. was grouped alone, but within the group of the species belong to the genus Origanum L. 13 Discussion The medicinal and aromatic plants have essential oils produced as secondary metabolites, which are used in the medicine and cosmetic industry, as antimicrobial in the food industry to inhibit the growth of microorganisms and for the food preservation [30]. It was aimed to investigate the potential antimicrobial activities of ten different species belonging to three different genera and their molecular characterizations using ISSR markers. The antibacterial activities of the extracts obtained from some species studied had differential effects on the bacterial strains. It was observed that the extracts obtained from all the species were more effective on the S. aureus than the other bacterial strains in general. According to pairwise comparisons, the bacterial strain S. aureus was the most sensitive strain, while P. aeruginosa was the most resistant strain in this study. The polyphenolic extracts of the medicinal herbs, which are able to change the membrane permeability and immediately access Molecular Biology Reports (2023) 50:289–298 295 Fig. 1 The ISSR band patterns produced with UBC-808 in the species analyzed in the study Fig. 2 The dendrogram based on the ISSR data displaying the phylogenetic relationships among the species analyzed in the study into the cell lead to the distruction of membrane structure and loss of function or death, have the differential inhibitory activities on Gr(+) and Gr(-) bacteria due to the differences in their cellular membranes [31, 32]. The inhibitory effects of plant extracts on Gr(+) bacteria were reported in the species; Origanum onites and Origanum vulgare ssp. hirtum [33], Origanum vulgare subsp. hirtum [34], Origanum vulgare [35], in the species belong to Lamiaceae and Astaraceae families [36], in the species Rosmarinus officinalis, Thymus capitatus, Origanum majorana and Salvia officinalis [37] in the previous studies and as well in this study. The different types of the secondary metabolites in plants might have been extracted with different solvents. Some species from the three genera studied had differential antimicrobial activities on the bacterial strains. This might be related to the types of solvents and those species might have differential antimicrobial active substances profiles as reported by Anastasakia et al. [36]. The effects of the antimicrobial activities of the ethanol [38–40] and chloroform extracts [41] were more effective than the hexane extracts on the bacterial strains in this study. It can be inferred that ethanol and chloroform could enhance the extraction of the secondary metabolites, which have more effective antimicrobial activities. The different medicinal herbs have different chemical compositions including different essential oils and polyphenolic compounds with different concentrations. The chemical compositions may even display variations depending on harvesting time and taxonomic classification [30] as observed in this study. All the species used in the study had strong antimicrobial activities according to 13 296 extraction with ethanol and chloroform, but they had relatively weak antimicrobial activities extraction with hexane. There were no significant differences on the bacterial strains according to application of different volumes of extracts in this study. When the overall results were evaluated, all the species belonging to the three genera differed significantly in their antimicrobial activities on the bacterial strains. The species even in the same genus might have different secondary metabolite profiles, which might have existed in different amounts. All the species analyzed in this study were originally collected from different locations and grown in the fields of GAP Institute. They have undergone genetic changes and adapted to the geographical and climatic conditions in their natural habitats. However, they continue to store information about the changes in their genomes. Therefore, the cultivation of all the species used in this study under the same environmental conditions minimized the effects of external conditions on the secondary metabolite diversity, concentration and amount of the plants. Thus, the variabilities in their antimicrobial activities were most probably corresponding to the variation in their genotypes. In the previous studies, the correlation between the concentrations of oil components and different types of molecular markers was performed to infer the linkage between the genes controlling the synthesis of secondary products and genotypes [8, 10]. The Pearson’s correlation values between some of the ISSR loci and antimicrobial activities of all the species on the bacterial strains displayed that there were strong positive and negative correlations between them. These correlations might be informative about defining the associations between the molecular markers and the essential oils or other secondary metabolites, which have antimicrobial activities. In further studies, using different marker systems could display the linkage between the markers and the genes controlling the production of essential oils pathway, and could be used for breeding studies with marker assisted selection. The results of the ISSR analysis indicated that the species had a very high level of genetic diversity. This is probably due to the fact that the species used in this study, except Origanum onites L., are wild species. Because wild relatives of cultivars contain high levels of genetic variation in their gene pool. By using different molecular marker systems, this genetic diversity can be used more efficiently in breeding studies by associating secondary metabolites with high antimicrobial and antioxidant properties. When the dendrogram was examined, the ISSR marker system clearly differentiated wild and cultivated species under study. It is clearly seen that Origanum onites, which is the only cultivated form, differed from other species in the Origanum L. genus and the wild species belonging to the Tyhmus L. and Thymbra L. genera. As a cultivated form, 13 Molecular Biology Reports (2023) 50:289–298 Origanum onites differentiated considerably from its wild relatives in terms of genetic structure due to it being grown under controlled environment compared to natural habitats where the wild relatives grew. Looking at the phylogenetic relationships of other species, Thymbra spicata L. appears to cluster with wild Origanum L. species. This shows that Thymbra spicata L. is closer to the species belonging to the genus Origanum than the species belonging to the genus Thymus L. in terms of genetic content. However, to claim this it is needed to analyze more samples from the species Thymbra spicata L. The Thymbra spicata L. species used in this study were collected from Mardin Darülzaferan and propagated in the trial field. Since Mardin is located in a sheltered area among the high mountains in the Southeastern Anatolia region, it has a milder climate than other provinces in the same region. The Origanum L. species used in this study were collected from temperate regions of Turkey, except for one. Climatic similarity in the environments where they grow may have been effective in preserving the common genetic structures of Thymbra spicata L. and Origanum L. species. The reason why Thymus cilicicus clustered separately from other Thymus L. species, which were clustered together, Thymus cilicicus was collected from the province of Kahramanmaraş, whereas the other two species were taken from the Yalova Ataturk Horticultural Center. Geographic and climatic differences accompanying the differences in their germplasm may be effective reasons for the differentiation of Thymus cilicicus L. from other species in the genus Thymus L. In addition, the provinces where the species taken from the Yalova Ataturk Horticultural Center were originally collected may be the same or close locations to each other, we do not have any information on this subject. Therefore, gene exchange between them may have continued probably in some way. Conclusion The ethanol and chloroform extracts of Thymus vulgaris L. and Thymbra spicata ssp. spicata L. were determined as the most effective species on both Gr(-) bacterial strains and Gr(+) bacterial strains and followed by the chloroform extracts of some species belonging to the genus Origanum. Ethanol and chloroform were effective solvents and could be used for extraction of essential oils in the genera of Thymus and Thymbra. Although hexane was observed to be an effective solvent for the species belonging to the genus Origanum L., the antimicrobial activities of the extracts were lower than those of ethanol and chloroform. Hexane extracts of the species Origanum vulgare L. ssp. hirtum and Origanum vulgare L. ssp. gracile indicated that they Molecular Biology Reports (2023) 50:289–298 had effective antimicrobial activities on both Gr(-) bacterial strains and Gr(+) bacterial strains. ISSR molecular markers showed high potential genetic diversity in all the species, and this genetic diversity also overlapped with the highlevel effects observed in antimicrobial activities. In order to develop commercial varieties with high antimicrobial and antioxidant activities and to make industrial production, it is necessary to determine the parents to be used in breeding studies. For this purpose, performing antimicrobial and antioxidant analyses on existing species and associating the obtained data with molecular markers form the basis of molecular breeding studies today. The results obtained in this study will also provide important data for further breeding studies in the future. 297 7. 8. 9. 10. 11. 12. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s11033022-07923-y. 13. Author contributions: ÖÖ and ASG designed the study. FT provided the plant samples, SK and NK performed the experiments and collected the data. ÖÖ wrote the manuscript and all the authors confirmed the final draft. Funding: This project (number FEF19002.19.001) is Sibel Kerem’s Master of Science thesis and was supported by Scientific Research Projects Unit (BAP) of Hitit University. Data Availability The datasets analyzed during the present study are available from the corresponding author on reasonable request. Declarations 14. 15. 16. 17. Conflict of interest: The authors declare that they have no conflict of interest. Ethical approval: The study was not involving Human Participants and/or Animals. 18. 19. References 1. 2. 3. 4. 5. 6. Simpson MG (2010) Plant Systematics, 2nd ed. 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA Davis PH (1982) Flora of Turkey and the East Aegean Islands, vol 7. Edinburgh University Press, Edinburgh, UK, pp 400–461 Ietswaart JH (1980) A Taxonomic Revision of the Genus Origanum (Labiatae). PhD Thesis, 4. Leiden University Press, The Hague, pp.153 Bakis Y, Babac MT, Uslu E (2011) “Updates and improvements of Turkish Plants Data Service (TÜBİVES)” In Health Informatics and Bioinformatics (HIBIT), 2011 6th International Symposium on (pp.136–140). IEEE. Morales R (2002) The history, botany and taxonomy of the genus Thymus. In: Stahl-Biskup E, Sáez F (eds) Thyme: The Genus Thymus. CRC Press Taylor and Francis Group, London, pp 1–43 Tohidia B, RahimmalekaM, Trindadeb H (2019) Review on essential oil, extracts composition, molecular and phytochemical properties of Thymus species in Iran. Ind Crops Prod 134:89–99 20. 21. 22. 23. Bozkurt İA, Soylu S, Kara M, Soylu EM (2020) Chemical Composition and Antibacterial Activity of Essential Oils Isolated from Medicinal Plants against Gall Forming Plant Pathogenic Bacterial Disease Agents. Kahramanmaras Sutcu Imam University J Agric Nat 23(6):1474–1482 Echeverrigaray S, Agostini G, Atti-Serfini L, Paroul N, Pauletti GF, Atti dos Santos AC (2001) Correlation between the Chemical and Genetic Relationships among Commercial Thyme Cultivars. J Agric Food Chem 49:4220–4223 György Z, Incze N, Pluháºẚr Z (2020) Differentiating Thymus vulgaris chemotypes with ISSR molecular markers. Biochem Syst Ecol 92:104–118 Rustaieea AR, Yavari A, Nazerib V, Shokrpour M, Sefidkonc F, Rasoulid M (2013) Genetic Diversity and Chemical Polymorphism of Some Thymus Species. Chem Biodiver 10:1088–1098 Godwin ID, Aitken EAB, Smith W (1997) Application of inter simple sequence repeat (ISSR) markers to plant genetics. Electrophoresis 18:1524–1528 Basha SD, Francis G, Makkar HSP, Becke K, Sujatha M (2009) A comparative study of biochemical traits and molecular markers for assessment of genetic relationships between Jatropha curcas L. germplasm from different countries. Plant Sci 176:812–823 Jin Z, Li J (2007) ISSR analysis on genetic diversity of endangered relic shrub Sinocalycanthus chinensis. Chin J Appl Ecol 18:247–253 Pivoriene O, Pasakinskiene I, Brazauskas G, Lideikyte L, Jensen LB, Lubberstedt T (2008) Inter simple sequence repeat (ISSR) loci mapping in the genome of perennial ryegrass. Biologia 54:17–21 Golkar P, Arzani A, Rezaei AM (2011) Genetic Variation in Safflower (Carthamus tinctorious L.) for Seed Quality-Related Traits and Inter-Simple Sequence Repeat (ISSR) Markers. Int J Mol Sci 12:2664–2677 Bornet BC, Muller FP, Branchard M (2002) Highly informative nature of inter simple sequence repeat (ISSR) sequences amplified using tri- and tetra-nucleotide primers from DNA of cauliflower (Brassica oleracea var. botrytus L.). Genome 45:890–896 El-Demerdash ES, Elsherbeny EA, Salama YAM, Ahmed MZ (2019) Genetic diversity analysis of some Egyptian Origanum and Thymus species using AFLP markers. J Genet Eng Biotechnol 7(1):13 Yousefi V, Najaphy A, Zebarjadi A, Safari H (2015) Molecular characterization of Thymus species using ISSR markers. J Anim Plant Sci 25(4):1087–1094 Karaca M, Ince AG, Aydin A, Elmasulu SY, Turgut K (2015) Microsatellites for genetic and taxonomic research on thyme (Thymus L.). Turk J Biol 39:147–159 Taşcıoğlua T, Sadıkoğlu N, Doğanlara S, Frary A (2018) Molecular genetic diversity in the Origanum genus: EST-SSR and SRAP marker analyses of the 22 species in eight sections that naturally occur in Turkey. Ind Crops Prod 123:746–761. doi:https://doi. org/10.1016/j.indcrop.2018.07.027 Ince AG, Karaca M, Elmasulu SY (2014) New microsatellite and CAPS-microsatellite markers for clarifying taxonomic and phylogenetic relationships within Origanum L. Mol Breeding 34:643–654 Katsiotis A, Nikoloudakis N, Linos A, Drossou A, Constantinidis T (2009) Phylogenetic relationships in Origanum spp. based on rDNA sequences and intra-genetic variation of of Greek O. vulgare subsp. hirtum revealed by RAPD. Scie Hortic 121(1):103– 108. doi:https://doi.org/10.1016/j.scienta.2009.01.015 Azizi A, Wagner C, Honermeier B, Friedt W (2009) Intraspecific diversity and relationship between subspecies of Origanum vulgare revealed by comparative AFLP and SAMPL marker analysis. Plant Syst Evol 281:151–160 13 298 24. da Silva DV, Duarte DV, Miguel JM, Leitão MG (2017) JM AFLP assessment of the genetic relationships among 12 Thymus taxa occurring in Portugal. Plant Genetic Resources: Characterization and Utilization 15(1): 89–92 25. El Sherbeny EA, El-Demerdash ES, Salama YAM, Ahmed MZS (2019) Genetic diversity among Thymus spp. Using RAPD and ISSR markers. Egypt J Desert Res 69:91–106 26. Hudzicki J (2009) Kirby-Bauer Disk. Diffusion Susceptibility Test Protocol 27. Kidwell KK, Osborn TC (1992) Simple plant DNA isolation procedures. In: Beckmann JS, Osborn TC (eds) Plant Genomes: Methods for Genetic and Physical Mapping. Springer, Dordrecht 28. Yeh FC, Yang RC, Boyle T, Ye ZH, Mao JX (1997) POPGENE (version 1.32), The user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Center University of Alberta Canada 26 29. Nei M (1973) Analysis of gene diversity in subdivided populations (population structure/genetic variability/heterozygosity/gene differentiation). Proc Natl Acad Sci USA part I 70(12):3321–3323 30. Stefanakis MK, Touloupakis E, Anastasopoulos E, Ghanotakis D, Katerinopoulos HE, Makridis P (2013) Antibacterial activity of essential oils from plants of the genus Origanum. Food Control 34(2):539–546. https://doi.org/10.1016/j.foodcont.2013.05.024 31. Davidson PM (1997) Chemical preservatives and natural antimicrobial compounds. In: Doyle MP, Beuchat LR, Montville TJ (eds) Food Microbiology Fundamentals and Frontiers. American Society for Microbiology, Washington, pp 520–556 32. Russo M, Suraci F, Postorino S, Serra D, Roccotelli A, Agosteo GE (2013) Essential oil chemical composition and antifungal effects on Sclerotium cepivorum of Thymus capitatus wild populations from Calabria, southern Italy. Rev Bras Farmacogn 23:239–248 33. Dortunç T, Cevikbaş A (1992) The investigations on the antibacterial and antifungal effects of some volatile oils. Marmara Pharm J 8(2):117–128 34. Esen G, Azaz AD, Kurkcuoglu M, Baser KHC, Tinmaz A (2007) Essential oil and antimicrobial activity of wild and cultivated Origanum vulgare L. subsp. hirtum (Link) Ietswaart from the Marmara region, Turkey. Flavour Fragr J 22:371–376 13 Molecular Biology Reports (2023) 50:289–298 35. Höferl M, Buchbauer G, Jirovetz L, Schmidt E, Stoyanova A, Denkova Z, Slavchev A, Geissler M (2009) Correlation of Antimicrobial Activities of Various Essential Oils and Their Main Aromatic Volatile Constituents. J Essent Oil Res 21(5):459–463 36. Anastasaki E, Zoumpopoulou G, Astraka K, Kampoli E, Skoumpi G, Papadimitriou K, Tsakalidou E, Polissiou M (2017) Phytochemical analysis and evaluation of the antioxidant and antimicrobial properties of selected herbs cultivated in Greece. Ind Crop Prod 108:616–628 37. Moumni S, Elaissi A, Trabelsi A, Merghni A, Chraief I, Jelassi B, Chemli R, Ferchichi S (2020) Correlation between chemical composition and antibacterial activity of some Lamiaceae species essential oils from Tunisia. BMC Complement Med Ther 20:103 38. Demirkol G, Ertürk Ö (2019) Antimicrobial and Antioxidant Effects of Spice Extracts. Turk J Agric Nat Sci 6(2):305–313 39. Yiğit D, Kandemir A (2002) Antimicrobial activity of some endemic plants (Salvia cryptantha, Origanum acutidens, Thymus sipyleus ssp. sipyleus). Erzincan Üni Eğ Fak Der, 4(2): 77–81 (in Turkish) 40. Ynalvez RA, Compean KL, Addo-Mensah A (2018) Qualitative Determination of the Secondary Metabolites and Evaluation of the Antimicrobial Activity of Leaf Extracts from Different Plant Families (Boraginaceae, Fabaceae, Lamiaceae and Lauraceae) against Microorganisms of Clinical Importance. J Pharm Res Int 23(2):1–12 41. Abu-Darwish MS, Al-Ramamneh EM, Kyslychenko VS, Karpiuk UV (2012) The antimicrobial activity of essential oils and extracts of some medicinal plants grown in Ash-shoubak region - South of Jordan. Pak J Pharm Sci 25(1):239–246 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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