The effect of isabelin, a sesquiterpene lactone from Ambrosia artemisiifolia on soil microorganisms and human pathogens

The effect of isabelin, a sesquiterpene lactone from Ambrosia artemisiifolia on soil... Abstract Ambrosia artemisiifolia L. (common ragweed) is an invasive weed, which is well known for the strong allergenic effect of its pollen as well as for its invasiveness and impact in crop fields (e.g. causing yield losses). This species produces a broad range of sesquiterpenoids. In recent years, new bioactive molecules have been discovered in this plant, e.g. isabelin, a sesquiterpene dilactone. The bioactivity of isabelin has been already demonstrated on allergy-related receptors and its inhibitory effect on seeds of various plant species. Isabelin was tested for potential antimicrobial effects by using a selection of soil-borne bacteria and fungi and three human pathogens as model organisms. For the majority of microorganisms tested, no antimicrobial activity of isabelin was observed. However, isabelin revealed strong antimicrobial activity against the Gram-positive soil bacterium Paenibacillus sp. and against the Gram-positive, multidrug-resistant Staphylococcus aureus. The observed inhibitory activity of isabelin can enlighten the importance to study similar compounds for their effect on human pathogens and on soil and rhizosphere microorganisms. Ambrosia artemisiifolia, isabelin, sesquiterpene, soil microorganism, human pathogens, antimicrobial activity INTRODUCTION Ambrosia artemisiifolia L. (Asteraceae) is a highly invasive weed, indigenous in North America and nowadays widespread through Europe and Asia (Kong, Wang and Xu 2007; Bullock et al.2010). The SMARTER European COST action (www.ragweed.eu) was initiated in the past years focusing on the sustainable management of this species, in particular due to its impact on human health. Problems caused by common ragweed are mainly related to strong allergenic responses to its abundant pollen, as well as interference in crops and invasiveness in urban areas (Casarini 2002; Genton, Shykoff and Giraud 2005; Kong, Wang and Xu 2007; Burbach et al.2009; Bullock et al.2010; Sang, Liu and Axmacher 2011; Chapman et al.2016). The Asteraceae are the main plants producing sesquiterpenoids, which are vast and diverse class of compounds with different functions, including the inhibition of other plant species and influence on the microbial community (Chadwick et al.2013). Ambrosia artemisiifolia produces a broad range of these molecules, and new compounds such as artemisiifolinic acid and isoartemisiifolinic acid have been reported in recent years (Taglialatela-Scafati et al.2012; Ding et al.2015). Metabolites of A. artemisiifolia have been already studied for their inhibitory potential on crops and weeds (Kong, Wang and Xu 2007; Vidotto, Tesio and Ferrero 2013; Molinaro et al.2016) and on different bacterial strains (Solujic et al.2008) including Staphylococcus aureus. Isabelin (Fig S1, Supporting Information) is a germacranolide sesquiterpene dilactone present in common ragweed extract (Seaman 1982). Its inhibitory activity as a pure compound on germination of seeds of different plant species has been recently observed (Molinaro et al.2016). Its structure, first identified in 1969 from A. psilostachya (Yoshioka and Mabry 1969), presents a lactone ring and a α-methylene-γ-lactone ring on the typical germacrene backbone. The presence of two different electrophilic Michael acceptor sites on C-13 and C-5 demonstrated to provide a higher reactivity compared to costunolide in previous studies about allergy-related receptors (Avonto et al.2011; Taglialatela-Scafati et al.2012). Ambrosia artemisiifolia has been studied for its invasiveness and escape attitude, showing an important effect on the soil biota by promotion and inhibition of growth, due to the previous presence of the same species (MacKay and Kotanen 2008). The morphological and physiological traits of invasive plant species have shown to alter soil nutrient cycles. However, these changes cannot occur without concomitant responses of the microbial community that reside in the rhizosphere (Hawkes et al.2005). Compounds released by the plant may affect soil microorganisms such as bacteria and fungi. However, very little is known about the direct effect and antimicrobial activity of compounds produced by A. artemisiifolia, such as isabelin, on soil microorganisms. In the past decades, the number of bacteria resistant to frontline antibiotics increased dramatically in comparison to the stagnating number of new antimicrobial compounds discovered for clinical use (Spellberg et al.2008). Plant extracts and essential oils contain many different compounds like sesquiterpenoids, which can exhibit antimicrobial activity. These sesquiterpenpoid compounds can be important in the fight against increasing antimicrobial resistance in human pathogens (Solujic et al.2008; Hristova et al.2015). Hence, the aims of this study were to test isabelin for antimicrobial activity (i) on a set of soil-borne fungi and bacteria and (ii) on three human pathogenic model organisms. MATERIAL AND METHODS Bacteria and culture conditions To test the effect of isabelin on soil bacteria, 10 phylogenetically different soil bacterial strains isolated from soils and sandy dune soils in The Netherlands were used: Achromobacter denitrificans sp. MH58.1, Arthrobacter sp. V13, Burkholderia sp. AD24, Collimonas sp. Ter91, Ochrobactrum sp. 44, Paenibacillus sp. AD87, Pseudomonas fluorescens sp. AD21, Serratia plymuthica PGPRI-I, Stenotrophomonas maltophilia sp. MH58.27 and Xanthomonas campestris sp. V79 (Czajkowski et al.2011; Garbeva et al.2014; Tyc et al.2017). The soil bacteria were pre-cultured from –80°C glycerol stocks on one-tenth Tryptone Soya Broth agar (Garbeva and de Boer 2009) and incubated for 3 days at 24°C prior application. To test the effect of isabelin on human pathogenic model bacterial strains, two indicator bacteria namely Escherichia coli WA321 (DSMZ # 4509) and S. aureus 533R4 (DSMZ # 20231) were used (Meyer and Schleifer 1978; Tyc et al.2014). The indicator bacteria were pre-cultured from –80°C glycerol stocks on LB-A medium (LB-Medium Lennox, Carl Roth GmbH + Co. KG, 20 g L–1 Merck Agar) and incubated overnight at 37°C prior application. All bacterial strains used in this study are listed in Table 1. Table 1. Bacterial, fungal and oomycetal organisms used in this study. Strain  Phylum/class  Genbank  Reference  Function  Fungal/oomycetal test organisms  Candida albicans BSMY 212 DSMZ 10697  Saccharomycetes  –  Schmidt (1996)    Chaetomium sp.  Ascomycota  –  Garbeva et al. (2014)    Fusarium culmorum PV  Ascomycota  –  Garbeva et al. (2014)    Fusarium oxysporum  Ascomycota  –  Garbeva et al. (2014)    Fusarium solani  Ascomycota  –  Garbeva et al. (2014)  Eukaryotic model organisms  Mucor hiemalis  Zygomycota  –  Garbeva et al. (2014)    Pythium ultimum P17  Oomycete  KT124638  Garbeva et al. (2014)    Rhizoctonia solani AG2.2IIIB  Basidiomycota  KT124637  Garbeva et al. (2014)    Ttichoderma harzianum  Ascomycota  –  Garbeva et al. (2014)    Verticillium albo-atrum  Ascomycota  –  Garbeva et al. (2014)    Bacterial test organisms  Achromobacter denitrificans sp. MH58.1  Actinobacteria  –  Garbeva et al. (2014)    Arthrobacter sp. V13  Actinobacteria  –  Garbeva et al. (2014)    Burkholderia sp. AD24  β-Proteobacteria  PRJNA320371  Tyc et al. (2017)    Collimonas sp. Ter91  β-Proteobacteria  –  Song et al. (2015)    Escherichia coli WA321 DSMZ 4509  γ-Proteobacteria  –  Tyc et al. (2014)    Ochrobactrum sp. 44  α-Proteobacteria  –  Czajkowski et al. (2011)  Bacterial model organisms  Paenibacillus sp. AD87  Firmictues  LXQN00000000  Tyc et al. (2017)    Pseudomonas fluorescence sp. AD21  γ-Proteobacteria  –  Garbeva et al. (2014)    Serratia plymuthica PGPRI-1  γ-Proteobacteria  –  Garbeva et al. (2014)    Staphylococcus aureus 533R4 Serovar 3 DSMZ 20231  Firmicutes  LN681573      Stenotrophomonas maltophilia sp. MH58.27  γ-Proteobacteria  –  Garbeva et al. (2014)    Xanthomonas campestris sp. V79  γ-Proteobacteria  –  Garbeva et al. (2014)    Strain  Phylum/class  Genbank  Reference  Function  Fungal/oomycetal test organisms  Candida albicans BSMY 212 DSMZ 10697  Saccharomycetes  –  Schmidt (1996)    Chaetomium sp.  Ascomycota  –  Garbeva et al. (2014)    Fusarium culmorum PV  Ascomycota  –  Garbeva et al. (2014)    Fusarium oxysporum  Ascomycota  –  Garbeva et al. (2014)    Fusarium solani  Ascomycota  –  Garbeva et al. (2014)  Eukaryotic model organisms  Mucor hiemalis  Zygomycota  –  Garbeva et al. (2014)    Pythium ultimum P17  Oomycete  KT124638  Garbeva et al. (2014)    Rhizoctonia solani AG2.2IIIB  Basidiomycota  KT124637  Garbeva et al. (2014)    Ttichoderma harzianum  Ascomycota  –  Garbeva et al. (2014)    Verticillium albo-atrum  Ascomycota  –  Garbeva et al. (2014)    Bacterial test organisms  Achromobacter denitrificans sp. MH58.1  Actinobacteria  –  Garbeva et al. (2014)    Arthrobacter sp. V13  Actinobacteria  –  Garbeva et al. (2014)    Burkholderia sp. AD24  β-Proteobacteria  PRJNA320371  Tyc et al. (2017)    Collimonas sp. Ter91  β-Proteobacteria  –  Song et al. (2015)    Escherichia coli WA321 DSMZ 4509  γ-Proteobacteria  –  Tyc et al. (2014)    Ochrobactrum sp. 44  α-Proteobacteria  –  Czajkowski et al. (2011)  Bacterial model organisms  Paenibacillus sp. AD87  Firmictues  LXQN00000000  Tyc et al. (2017)    Pseudomonas fluorescence sp. AD21  γ-Proteobacteria  –  Garbeva et al. (2014)    Serratia plymuthica PGPRI-1  γ-Proteobacteria  –  Garbeva et al. (2014)    Staphylococcus aureus 533R4 Serovar 3 DSMZ 20231  Firmicutes  LN681573      Stenotrophomonas maltophilia sp. MH58.27  γ-Proteobacteria  –  Garbeva et al. (2014)    Xanthomonas campestris sp. V79  γ-Proteobacteria  –  Garbeva et al. (2014)    View Large Eukaryotic target organisms and culture conditions To detect antifungal activity of isabelin, eight fungal model organisms isolated from a sandy dune soil in The Netherlands were used: Chaetomium sp., Fusarium culmorum PV, F. oxysporum, F. solani, Mucor hiemalis, Rhizoctonia solani AG2.2 IIIB, Trichoderma harzianum, Verticillium albo-atrum and one oomycete: Pythium ultimum P17 (de Rooij-van der Goes, van der Putten and van Dijk 1995; Garbeva et al.2014). The fungi and the oomycete were pre-cultured on one-fifth Potato Dextrose Agar (29 g L−1 Oxoid CM 139) (Fiddaman and Rossall 1993) and incubated at 24°C for 5 days prior application. As a model organism for yeast-like fungi, the yeast Candida albicans BSMY 212 (DSMZ # 10697) (Schmidt 1996) was used. The yeast was pre-cultured from –80°C glycerol stocks on YEPD plates (20 g L−1 Merck Dextrose, 20.0 g L−1 BACTO Peptone, 10.0 g L−1 BACTO Yeast extract, 20 g L−1 Merck Agar) and incubated at 37°C. All used model organisms are listed in Table 1. Isabelin purification and preparation of stock solution All used reagents were analytical or LC-MS grade and were obtained from Sigma-Aldrich, Milan, Italy. Ambrosia artemisiifolia plant material was collected in University of Turin Campus (Agriculture, Forest and Food Science Department, Grugliasco (TO), 45°03'58.8‘N 7°35'36.3'E) from invasive colonies next to maize and soybean fields in July 2016. For the methanol extraction, fractionation of the A. artemisiifolia extract and the preparation of pure isabelin, the previously developed semipreparative LC method was used (Molinaro et al.2016). For each preparative injection, 1 mL of methanol concentrated extract was diluted with 1 mL of MilliQ water and the total 2 mL was injected using a 2-mL loop on a Rheodyne® injection valve. Pure isabelin fractions were obtained using a semipreparative LC column (GL Sciences C18, 10 × 150 mm, Milan, Italy) in isocratic method with 70% H2O, 30% acetonitrile and a total flow of 4 mL/min. In the expected retention time (20–25 min), 1-min fractions (4 mL each one) were collected. Each fraction was checked through LC-MS in order to assure the purity of isabelin. The LC-MS system was a Varian MS-310 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source and 212 LC pump (Agilent, Milan, Italy). Separation was performed on a Kinetex RP C18 column, 5 μm particle size, 50 × 2.0 mm (Phenomenex, Torrance, CA, USA). The mobile phase was (A) water and (B) acetonitrile, both containing 0.1% (v/v) acetic acid. The mobile phase gradient was from 90% to 10% A in 10 min (0.2 mL/min flow rate). ESI conditions used in negative polarization mode for the full-scan method were as follows: needle potential –3500 V, shield –500 V and capillary –30 V. Gas conditions were set with 20.0 psi for nebulizing gas and 25.0 psi of N2 at 300°C as drying gas. The fractions containing pure isabelin were reunited, and the solvent was evaporated to dryness in order to obtain pure isabelin. The 1000 μg/mL stock solution was prepared by weighing isabelin and dissolving it in the corresponding amount of solvent (75% v/v acetonitrile or sterile demi water). Antibacterial and anti-yeast assays based on agar-diffusion test To test for antimicrobial activity against bacteria and the yeast, agar disk-diffusion tests were performed in 12-well plates (Balouiri, Sadiki and Ibnsouda 2016). Single colonies of E. coli WA321, S. aureus 533R4 and C. albicans BSMY 212 were picked from plates and grown overnight in liquid LB or YEPD media at 37°C, 220 rpm. Fresh LB agar or YEPD agar (1.0% Merck Agar) was prepared and cooled down to ∼45°C. The target organisms were added to a final OD600 of 0.002 corresponding to 6.0 × 105 CFU/mL (E. coli WA321), 4.0 × 105 CFU/mL (S. aureus 533R4) or 1.6 × 104 cells/mL (C. albicans BSMY 212). A volume of 1 mL of seeded agar was added to each well of the 12-well plates (Greiner bio-one, Cat# 665180). After solidification, a filter paper (diameter ∼5.5 mm) (Whatman, Cat# 1003–150, 6 μm pore size) was added on the top of the agar surface and a volume of 5 μl of 1000 μg/mL isabelin (=5 μg) was added to the filter paper. As control, 5 μl of the solvent (75% acetonitrile) was added. Additionally, for positive assay control, 5 μl of appropriate antibiotic (ampicillin 100 mg/mL for E. coli WA321, tetracycline 15 mg/mL for S. aureus 533R4 or cycloheximide 25 mg/mL for C. albicans BSMY 212) was used. As negative assay control, filter papers without added antibiotics or isabelin were applied. The plates were incubated overnight at 37°C and on the next day plates were examined for visible zones of inhibition (ZOI) around the filter papers. Digital photographs were taken and analyzed using the AXIO VISION v4.8 imaging Software (Carl Zeiss Imaging Solutions GmbH) for surface-area determination (in pixel2). All treatments were performed in quadruplicates. Bioassays to test for antifungal/anti-oomycetal activity based on agar-diffusion test To test the effect of isabelin on fungal and oomycetal growth (mycelial extension), the following fungi/oomycetes were used: Chaetomium sp., F. culmorum PV, F. oxysporum, F. solani, M. hiemalis, P. ultimum P17, R. solani AG2.2 IIIB, T. harzianum, V. albo-atrum. The assays were performed in 12-well plates (Greiner bio-one, Cat# 665180). For testing the effect of isabelin on mycelial growth, fresh PD agar (29 g L−1 Oxoid CM 139) was prepared and a volume of 1 mL PD agar was added to each well. After solidification, the target fungi/oomycete were added by placing a 5-mm diameter plug of each fungus/oomycete at the top edge of each well. A filter paper with a diameter of ∼5.5 mm (Whatman, Cat# 1003–150, 6 μm pore size) was placed on the agar surface at the lower edge of each compartment, and a volume of 5 μl stock solution (corresponding to 5 μg) isabelin was added to each filter paper. For control, 5 μl of the solvent (75% v/v acetonitrile) was used. Plates were incubated at 24°C for 4 days. After incubation, plates were examined for fungal growth (=mycelial extension) and digital photographs were taken. The digital images were analyzed using the AXIO VISION v4.8 imaging Software (Carl Zeiss Imaging Solutions GmbH) to compare the growth of the treatments to the controls without added isabelin. In vitro test of growth inhibition on soil bacteria in liquid media For the determination of antimicrobial activity of isabelin in liquid media optical density (absorbance), measurements and counting of colony forming units (CFU) were performed in 96-well plates (Greiner bio-one, Cat# 655180). For the in vitro test, six phylogenetically different soil bacteria were used: A. denitrificans sp. MH58.1, Ps. fluorescences sp. AD21, Se. plymuthica sp. PGPRI-1, St. maltophilia sp. MH58.27, X. campestris sp. V79 and Paenibacillus sp. AD87. Single colonies of each bacterial isolate were picked from plates and grown in 20 mL of one-tenth Tryptic Soy Broth (TSB) overnight at 24°C, 200 rpm. For the absorbance measurement, 20 μl of 1000 μg/mL isabelin dissolved in water (10% v/v) (=20 μg) was dispensed in 180 μl of one-tenth TSB containing the soil bacterial isolates diluted to OD600 of 0.05. As positive assay control, 5 μl of 15 mg/mL of tetracycline (=75 μg) (Sigma-Aldrich product T-7660) was applied. The growth rates were monitored in the presence of isabelin and were compared to the growth rates of the controls (soil bacteria in the absence of isabelin) using a BioTek Synergy HT Multi-Mode Microplate Reader (Beun de Ronde Life Sciences, The Netherlands). The absorbance was measured using the following settings: measure absorbance at 600 nm, 24 h of measurement with an interval of 1 h, delay between each sample measurement was set to 50 ms, shaking intensity was set to 1 and the duration to 15 min before each measurement. The entire assay was performed at 24°C for 24 h. All treatments were measured in three replicates. The measured values were normalized against the mean absorbance values of the used culture media. In vitro test of growth inhibition on human pathogenic bacteria and yeast model organism The effect of pure isabelin was tested on three human pathogenic model organisms. For this, optical density measurements and counting of CFU were performed. Single colonies of each bacterial target organism, E. coli WA321, S. aureus 533R4 and of the yeast like fungi C. albicans BSMY 212, were picked from plates and grown overnight in 20 mL of LB broth or YEPD broth at 37°C, 220 rpm. Fresh LB or YEPD growth media was supplemented with the target organisms at a final OD600 of 0.002 corresponding to 6.0 × 105 CFU/mL (E. coli WA321), 4.0 × 105 CFU/mL (S. aureus 533R4) or 1.6 × 104 cells/mL (C. albicans BSMY 212). The assay was performed in 96-well plates (Greiner bio-one, Cat# 655180). For the measurement, 20 μl of 1000 ppm (equivalent to 20 μg) isabelin was dispensed in 180 μl of one-tenth LB or YEPD media containing the target organisms. As positive assay control, 5 μl of 15 mg/mL (=75 μg) of tetracycline (Sigma-Aldrich product T-7660) or 20 μl of 200 mg/mL (=4 mg) cycloheximide (Sigma-Aldrich) was applied. The growth rate of the model organisms was monitored in the presence of isabelin and was compared to the growth rates of the controls (in the absence of isabelin). The absorbance measurements were performed on a BioTek Synergy HT Multi-Mode Microplate Reader (Beun de Ronde Life Sciences, The Netherlands) with the following settings: measure absorbance at 600 nm, 24 h of measurement with 1-h interval, delay between each sample measurement 50 ms, shaking intensity 1 and 15 min between each measurement. The entire assay was performed at 37°C for 24 h. All treatments were measured in three replicates. The measured values were normalized against the mean absorbance values of the used culture media. Enumeration of target organism growth The growth of the target organisms grown in liquid was evaluated by plate counting. After 24 h of incubation and the optical density measurements, a sample of 100 μl of each well was taken and added to 900 μl of 10 mM phosphate buffer (KH2PO4, pH 6.5). Dilution series of each treatment were prepared in triplicates. A volume of 100 μl of each serial dilution was plated in three replicates with a disposable Drigalski spatula on one-tenth TSBA plates (soil bacteria), LB agar plates (human pathogenic model bacteria) or on YEPD agar plates (yeast model organism). The plates were incubated for 3 days at 24°C or overnight at 37°C, and CFU enumeration was carried out on an aCOlyte Colony Counter (Don Whitley Scientific, Meintrup DWS Laborgeräte GmbH, Germany). Statistical analysis Statistical analyses on fungal mycelial extension, the size of the ZOI and on viable count data (CFU) of bacteria and yeast-like fungi were performed with IBM SPSS Statistics 24 (IBM, Somers, NY, USA) using one-way ANOVA and post-hoc Tukey test (HSD) or two-tailed t-test between the data sets. Data were normalized by log transformation prior statistical analysis. The 5% level was taken as threshold for significance between the control and the treatments. RESULTS AND DISCUSSION Effect of isabelin on soil microorganisms Studying how plants and soil microorganisms interact is key to understand how plants can modify the soil environment by promoting or inhibiting specific soil microorganisms. Here we performed bioassays on commonly occurring soil microorganisms (fungi and bacteria) in order to determine potential growth inhibition or growth promotion by isabelin, a germacranolide sesquiterpene dilactone present in common ragweed extract, which has phytotoxic activity (Molinaro et al.2016). No changes in growth of fungi or oomycete could be observed in the presence of isabelin (Fig. 1). All tested soil fungi and the oomycete did not show significant differences of mycelial extension of the treatments compared to the controls. Figure 1. View largeDownload slide Effect of 5 μg isabelin dissolved in water (isabelin), water control (CTRL) on the growth of soil fungi and oomycetes. Effect on mycelial extension on (a) F. culmorum, (b) R. solani, (c) M. hiemalis and (d) P. ultimum. Data represent the mean of three replicates; error bars represent standard deviation. Figure 1. View largeDownload slide Effect of 5 μg isabelin dissolved in water (isabelin), water control (CTRL) on the growth of soil fungi and oomycetes. Effect on mycelial extension on (a) F. culmorum, (b) R. solani, (c) M. hiemalis and (d) P. ultimum. Data represent the mean of three replicates; error bars represent standard deviation. Although we did not observed any antifungal or anti-oomycetal activity exhibited by isabelin, which can be excreted by the invasive plant species A. artemisiifolia, other studies have demonstrated that invasive plant species are able to suppress arbuscular mycorrhizal fungi (Klironomos 2002; Callaway et al.2008; Rout and Callaway 2012). Bacterial enumeration of CFU after 24 h of growth in the presence of isabelin revealed the inhibition of only one of the tested soil bacteria, Paenibacillus sp. AD87 (Firmicutes) (Fig. 2). The growth of the Gram-positive Paenibacillus sp. AD87 was significantly inhibited (P < 0.001) by reaching 2.38 × 106 CFU/mL in presence of 20 μg isabelin compared to 2.64 × 107 CFU/mL in the control (Fig. 2). No effect by the presence of isabelin was observed on the other tested soil bacteria (A. denitrificans sp. MH58.1, Arthrobacter sp. V13, Burkholderia sp. AD24, Collimonas sp. Ter91, Ochrobactrum sp. 44, Ps. fluorescens sp. AD21, Se. plymuthica PGPRI-I, St. maltophilia sp. MH58.27, X. campestris sp. V79), belonging to the phyla of Actinobacteria, alpha-, beta- and gammaproteobacteria (Fig. S2, Table S1, Supporting Information). The genus Paenibacillus comprises bacterial species relevant to plants, rhizosphere and the soil environment. Many Paenibacillus species can promote crop growth directly via biological nitrogen fixation, phosphate solubilization, production of the phytohormone indole-3-acetic acid and the release of siderophores that enable iron acquisition. Furthermore, they can offer plant protection against insect herbivores and phytopathogens, including bacteria, fungi, nematodes and viruses (Ryu et al.2005; Anand, Grayston and Chanway 2013; Debois et al.2013). The observed specific effect of isabelin on the Paenibacillus genus may be providing a strategy of invasive plant to (i) inhibit this particular group of plant growth promoting bacteria (PGPR) and create an unfavorable environment for competitor plant species or (ii) to select specific microbiome for surrounding the roots of invasive plant species. Figure 2. View largeDownload slide CFUs of Paenibacillus sp. AD87 in the presence of 20 μg isabelin dissolved in water (isabelin), water control (CTRL) and 75 μg tetracycline as inhibition control (TC). Significant differences between treatments and the control are marked with an asterisk and the respective P-value. Data represent the mean of three replicates; error bars represent standard deviation. Figure 2. View largeDownload slide CFUs of Paenibacillus sp. AD87 in the presence of 20 μg isabelin dissolved in water (isabelin), water control (CTRL) and 75 μg tetracycline as inhibition control (TC). Significant differences between treatments and the control are marked with an asterisk and the respective P-value. Data represent the mean of three replicates; error bars represent standard deviation. Effect of isabelin on human pathogens Both the agar diffusion tests and liquid media bioassay on the human pathogens model organisms revealed inhibition on two of the three tested model species (Fig. 3). No inhibition was observed in the assays performed with E. coli. On the other hand, results obtained with S. aureus revealed a significant growth inhibition (P < 0.001) by reaching 1.33 × 106 CFU/mL in the presence of 20 μg isabelin compared to 8.44 × 108 CFU/mL in the control (Fig 3). Furthermore, the presence of 20 μg isabelin reduced absorbance values in OD600 measurement. This observation confirmed the appearance of the inhibition area (ZOI) in the agar-diffusion test. This clear difference in antimicrobial activity of isabelin observed against Gram-positive and Gram-negative bacteria is in line with other studies showing that Gram-positive bacteria are more sensitive to antimicrobial compounds than Gram-negative bacteria (Rice 2006; Giske et al.2008). Figure 3. View largeDownload slide Effect of 20 μg isabelin dissolved in water on the growth of three human pathogens: (a) E. coli WA321, (b) S. aureus 533R4, (c) C. albicans BSMY212. Controls: water control (CTRL) and 75 μg tetracycline (TC) or 4 mg Cycloheximide (CH) as inhibition control. Significant differences between treatments and the controls are marked with an asterisk and the respective P-value. Data represent the mean of three replicates; error bars represent standard deviation. Figure 3. View largeDownload slide Effect of 20 μg isabelin dissolved in water on the growth of three human pathogens: (a) E. coli WA321, (b) S. aureus 533R4, (c) C. albicans BSMY212. Controls: water control (CTRL) and 75 μg tetracycline (TC) or 4 mg Cycloheximide (CH) as inhibition control. Significant differences between treatments and the controls are marked with an asterisk and the respective P-value. Data represent the mean of three replicates; error bars represent standard deviation. The yeast-model organism, C. albicans, showed a significant (P = 0.001) decreased number of CFUs in the presence of 20 μg isabelin. Candida albicans reached 1.32 × 107 CFU/mL in the presence of 20 μg isabelin compared to 5.79 × 107 CFU/mL in the control (Fig. 3). This result is in accordance with the observed reduced absorbance values in the OD600 measurements. Candida species are ubiquitous common commensal yeasts, which are responsible for many nosocomial infections. Candida albicans, in particular, is responsible for more than half of all the candidemia cases (Lass-Flörl 2009). In the last decade, many Candida strains developed resistance to the azoles antibiotics, which are normally used for the treatment of these infections (Cleveland et al.2015). There is an increased interest in finding new active molecules with the ability of inhibiting Candida strains (Bona et al.2016). The cell wall organization in Gram-positive bacteria and Candida could possibly explain the observed growth-inhibitory effects of isabelin. Another sesquiterpene lactone, cnicin, a germacranolide sesquiterpene lactone found in Centauria species, is a molecule presenting Michael acceptors sites. Cnicin has been able to alkylate the thiol-groups of cysteine residue of the UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme involved in the peptidoglycan biosynthesis, causing the inhibition of the biosynthesis of the cell wall (Bachelier, Mayer and Klein 2006). The high reactivity of isabelin with thiol groups has been already demonstrated (Taglialatela-Scafati et al.2012), and the action on cell wall biosynthesis could be responsible for the antimicrobial activity of this compound. CONCLUSIONS This is the first study to test the antimicrobial activity of the pure sesquiterpene dilactone isabelin, produced by the invasive weed A. artemisiifolia, against a range of phylogenetically different soil-borne microorganisms and human pathogens. Isabelin did not reveal any antifungal or anti-oomycetal activity; however, it revealed strong antimicrobial activity against the Gram-positive soil bacteria Paenibacillus sp., the multidrug-resistant human pathogen S. aureus and the yeast human pathogen C. albicans. Many sesquiterpene lactones have been related to allergenic effects; however, little is known to date about the antimicrobial properties of these molecules. Our results indicate that isabelin can inhibit certain soil borne bacteria and hence, modify the surrounding soil microbiome. Moreover, isabelin revealed strong antimicrobial activity against important human pathogens, indicating that metabolites released by invasive plant species can be explored as a source of novel compounds needed to combat the raising multidrug-resistant pathogens. Further studies will be needed to explain the mechanisms of antimicrobial activity of isabelin as well as to examine possible link between allergenic effects and antimicrobial activity of this compound. SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online. Acknowledgements The authors would like to thank the two anonymous reviewers for their suggestion for improving the manuscript. This is publication 6380 of the NIOO-KNAW. Conflict of interests. None declared. REFERENCES Anand R, Grayston S, Chanway C. 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(compositae). Tetrahedron  1969; 25: 4767– 79. Google Scholar CrossRef Search ADS   © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Microbiology Letters Oxford University Press

The effect of isabelin, a sesquiterpene lactone from Ambrosia artemisiifolia on soil microorganisms and human pathogens

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Abstract

Abstract Ambrosia artemisiifolia L. (common ragweed) is an invasive weed, which is well known for the strong allergenic effect of its pollen as well as for its invasiveness and impact in crop fields (e.g. causing yield losses). This species produces a broad range of sesquiterpenoids. In recent years, new bioactive molecules have been discovered in this plant, e.g. isabelin, a sesquiterpene dilactone. The bioactivity of isabelin has been already demonstrated on allergy-related receptors and its inhibitory effect on seeds of various plant species. Isabelin was tested for potential antimicrobial effects by using a selection of soil-borne bacteria and fungi and three human pathogens as model organisms. For the majority of microorganisms tested, no antimicrobial activity of isabelin was observed. However, isabelin revealed strong antimicrobial activity against the Gram-positive soil bacterium Paenibacillus sp. and against the Gram-positive, multidrug-resistant Staphylococcus aureus. The observed inhibitory activity of isabelin can enlighten the importance to study similar compounds for their effect on human pathogens and on soil and rhizosphere microorganisms. Ambrosia artemisiifolia, isabelin, sesquiterpene, soil microorganism, human pathogens, antimicrobial activity INTRODUCTION Ambrosia artemisiifolia L. (Asteraceae) is a highly invasive weed, indigenous in North America and nowadays widespread through Europe and Asia (Kong, Wang and Xu 2007; Bullock et al.2010). The SMARTER European COST action (www.ragweed.eu) was initiated in the past years focusing on the sustainable management of this species, in particular due to its impact on human health. Problems caused by common ragweed are mainly related to strong allergenic responses to its abundant pollen, as well as interference in crops and invasiveness in urban areas (Casarini 2002; Genton, Shykoff and Giraud 2005; Kong, Wang and Xu 2007; Burbach et al.2009; Bullock et al.2010; Sang, Liu and Axmacher 2011; Chapman et al.2016). The Asteraceae are the main plants producing sesquiterpenoids, which are vast and diverse class of compounds with different functions, including the inhibition of other plant species and influence on the microbial community (Chadwick et al.2013). Ambrosia artemisiifolia produces a broad range of these molecules, and new compounds such as artemisiifolinic acid and isoartemisiifolinic acid have been reported in recent years (Taglialatela-Scafati et al.2012; Ding et al.2015). Metabolites of A. artemisiifolia have been already studied for their inhibitory potential on crops and weeds (Kong, Wang and Xu 2007; Vidotto, Tesio and Ferrero 2013; Molinaro et al.2016) and on different bacterial strains (Solujic et al.2008) including Staphylococcus aureus. Isabelin (Fig S1, Supporting Information) is a germacranolide sesquiterpene dilactone present in common ragweed extract (Seaman 1982). Its inhibitory activity as a pure compound on germination of seeds of different plant species has been recently observed (Molinaro et al.2016). Its structure, first identified in 1969 from A. psilostachya (Yoshioka and Mabry 1969), presents a lactone ring and a α-methylene-γ-lactone ring on the typical germacrene backbone. The presence of two different electrophilic Michael acceptor sites on C-13 and C-5 demonstrated to provide a higher reactivity compared to costunolide in previous studies about allergy-related receptors (Avonto et al.2011; Taglialatela-Scafati et al.2012). Ambrosia artemisiifolia has been studied for its invasiveness and escape attitude, showing an important effect on the soil biota by promotion and inhibition of growth, due to the previous presence of the same species (MacKay and Kotanen 2008). The morphological and physiological traits of invasive plant species have shown to alter soil nutrient cycles. However, these changes cannot occur without concomitant responses of the microbial community that reside in the rhizosphere (Hawkes et al.2005). Compounds released by the plant may affect soil microorganisms such as bacteria and fungi. However, very little is known about the direct effect and antimicrobial activity of compounds produced by A. artemisiifolia, such as isabelin, on soil microorganisms. In the past decades, the number of bacteria resistant to frontline antibiotics increased dramatically in comparison to the stagnating number of new antimicrobial compounds discovered for clinical use (Spellberg et al.2008). Plant extracts and essential oils contain many different compounds like sesquiterpenoids, which can exhibit antimicrobial activity. These sesquiterpenpoid compounds can be important in the fight against increasing antimicrobial resistance in human pathogens (Solujic et al.2008; Hristova et al.2015). Hence, the aims of this study were to test isabelin for antimicrobial activity (i) on a set of soil-borne fungi and bacteria and (ii) on three human pathogenic model organisms. MATERIAL AND METHODS Bacteria and culture conditions To test the effect of isabelin on soil bacteria, 10 phylogenetically different soil bacterial strains isolated from soils and sandy dune soils in The Netherlands were used: Achromobacter denitrificans sp. MH58.1, Arthrobacter sp. V13, Burkholderia sp. AD24, Collimonas sp. Ter91, Ochrobactrum sp. 44, Paenibacillus sp. AD87, Pseudomonas fluorescens sp. AD21, Serratia plymuthica PGPRI-I, Stenotrophomonas maltophilia sp. MH58.27 and Xanthomonas campestris sp. V79 (Czajkowski et al.2011; Garbeva et al.2014; Tyc et al.2017). The soil bacteria were pre-cultured from –80°C glycerol stocks on one-tenth Tryptone Soya Broth agar (Garbeva and de Boer 2009) and incubated for 3 days at 24°C prior application. To test the effect of isabelin on human pathogenic model bacterial strains, two indicator bacteria namely Escherichia coli WA321 (DSMZ # 4509) and S. aureus 533R4 (DSMZ # 20231) were used (Meyer and Schleifer 1978; Tyc et al.2014). The indicator bacteria were pre-cultured from –80°C glycerol stocks on LB-A medium (LB-Medium Lennox, Carl Roth GmbH + Co. KG, 20 g L–1 Merck Agar) and incubated overnight at 37°C prior application. All bacterial strains used in this study are listed in Table 1. Table 1. Bacterial, fungal and oomycetal organisms used in this study. Strain  Phylum/class  Genbank  Reference  Function  Fungal/oomycetal test organisms  Candida albicans BSMY 212 DSMZ 10697  Saccharomycetes  –  Schmidt (1996)    Chaetomium sp.  Ascomycota  –  Garbeva et al. (2014)    Fusarium culmorum PV  Ascomycota  –  Garbeva et al. (2014)    Fusarium oxysporum  Ascomycota  –  Garbeva et al. (2014)    Fusarium solani  Ascomycota  –  Garbeva et al. (2014)  Eukaryotic model organisms  Mucor hiemalis  Zygomycota  –  Garbeva et al. (2014)    Pythium ultimum P17  Oomycete  KT124638  Garbeva et al. (2014)    Rhizoctonia solani AG2.2IIIB  Basidiomycota  KT124637  Garbeva et al. (2014)    Ttichoderma harzianum  Ascomycota  –  Garbeva et al. (2014)    Verticillium albo-atrum  Ascomycota  –  Garbeva et al. (2014)    Bacterial test organisms  Achromobacter denitrificans sp. MH58.1  Actinobacteria  –  Garbeva et al. (2014)    Arthrobacter sp. V13  Actinobacteria  –  Garbeva et al. (2014)    Burkholderia sp. AD24  β-Proteobacteria  PRJNA320371  Tyc et al. (2017)    Collimonas sp. Ter91  β-Proteobacteria  –  Song et al. (2015)    Escherichia coli WA321 DSMZ 4509  γ-Proteobacteria  –  Tyc et al. (2014)    Ochrobactrum sp. 44  α-Proteobacteria  –  Czajkowski et al. (2011)  Bacterial model organisms  Paenibacillus sp. AD87  Firmictues  LXQN00000000  Tyc et al. (2017)    Pseudomonas fluorescence sp. AD21  γ-Proteobacteria  –  Garbeva et al. (2014)    Serratia plymuthica PGPRI-1  γ-Proteobacteria  –  Garbeva et al. (2014)    Staphylococcus aureus 533R4 Serovar 3 DSMZ 20231  Firmicutes  LN681573      Stenotrophomonas maltophilia sp. MH58.27  γ-Proteobacteria  –  Garbeva et al. (2014)    Xanthomonas campestris sp. V79  γ-Proteobacteria  –  Garbeva et al. (2014)    Strain  Phylum/class  Genbank  Reference  Function  Fungal/oomycetal test organisms  Candida albicans BSMY 212 DSMZ 10697  Saccharomycetes  –  Schmidt (1996)    Chaetomium sp.  Ascomycota  –  Garbeva et al. (2014)    Fusarium culmorum PV  Ascomycota  –  Garbeva et al. (2014)    Fusarium oxysporum  Ascomycota  –  Garbeva et al. (2014)    Fusarium solani  Ascomycota  –  Garbeva et al. (2014)  Eukaryotic model organisms  Mucor hiemalis  Zygomycota  –  Garbeva et al. (2014)    Pythium ultimum P17  Oomycete  KT124638  Garbeva et al. (2014)    Rhizoctonia solani AG2.2IIIB  Basidiomycota  KT124637  Garbeva et al. (2014)    Ttichoderma harzianum  Ascomycota  –  Garbeva et al. (2014)    Verticillium albo-atrum  Ascomycota  –  Garbeva et al. (2014)    Bacterial test organisms  Achromobacter denitrificans sp. MH58.1  Actinobacteria  –  Garbeva et al. (2014)    Arthrobacter sp. V13  Actinobacteria  –  Garbeva et al. (2014)    Burkholderia sp. AD24  β-Proteobacteria  PRJNA320371  Tyc et al. (2017)    Collimonas sp. Ter91  β-Proteobacteria  –  Song et al. (2015)    Escherichia coli WA321 DSMZ 4509  γ-Proteobacteria  –  Tyc et al. (2014)    Ochrobactrum sp. 44  α-Proteobacteria  –  Czajkowski et al. (2011)  Bacterial model organisms  Paenibacillus sp. AD87  Firmictues  LXQN00000000  Tyc et al. (2017)    Pseudomonas fluorescence sp. AD21  γ-Proteobacteria  –  Garbeva et al. (2014)    Serratia plymuthica PGPRI-1  γ-Proteobacteria  –  Garbeva et al. (2014)    Staphylococcus aureus 533R4 Serovar 3 DSMZ 20231  Firmicutes  LN681573      Stenotrophomonas maltophilia sp. MH58.27  γ-Proteobacteria  –  Garbeva et al. (2014)    Xanthomonas campestris sp. V79  γ-Proteobacteria  –  Garbeva et al. (2014)    View Large Eukaryotic target organisms and culture conditions To detect antifungal activity of isabelin, eight fungal model organisms isolated from a sandy dune soil in The Netherlands were used: Chaetomium sp., Fusarium culmorum PV, F. oxysporum, F. solani, Mucor hiemalis, Rhizoctonia solani AG2.2 IIIB, Trichoderma harzianum, Verticillium albo-atrum and one oomycete: Pythium ultimum P17 (de Rooij-van der Goes, van der Putten and van Dijk 1995; Garbeva et al.2014). The fungi and the oomycete were pre-cultured on one-fifth Potato Dextrose Agar (29 g L−1 Oxoid CM 139) (Fiddaman and Rossall 1993) and incubated at 24°C for 5 days prior application. As a model organism for yeast-like fungi, the yeast Candida albicans BSMY 212 (DSMZ # 10697) (Schmidt 1996) was used. The yeast was pre-cultured from –80°C glycerol stocks on YEPD plates (20 g L−1 Merck Dextrose, 20.0 g L−1 BACTO Peptone, 10.0 g L−1 BACTO Yeast extract, 20 g L−1 Merck Agar) and incubated at 37°C. All used model organisms are listed in Table 1. Isabelin purification and preparation of stock solution All used reagents were analytical or LC-MS grade and were obtained from Sigma-Aldrich, Milan, Italy. Ambrosia artemisiifolia plant material was collected in University of Turin Campus (Agriculture, Forest and Food Science Department, Grugliasco (TO), 45°03'58.8‘N 7°35'36.3'E) from invasive colonies next to maize and soybean fields in July 2016. For the methanol extraction, fractionation of the A. artemisiifolia extract and the preparation of pure isabelin, the previously developed semipreparative LC method was used (Molinaro et al.2016). For each preparative injection, 1 mL of methanol concentrated extract was diluted with 1 mL of MilliQ water and the total 2 mL was injected using a 2-mL loop on a Rheodyne® injection valve. Pure isabelin fractions were obtained using a semipreparative LC column (GL Sciences C18, 10 × 150 mm, Milan, Italy) in isocratic method with 70% H2O, 30% acetonitrile and a total flow of 4 mL/min. In the expected retention time (20–25 min), 1-min fractions (4 mL each one) were collected. Each fraction was checked through LC-MS in order to assure the purity of isabelin. The LC-MS system was a Varian MS-310 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source and 212 LC pump (Agilent, Milan, Italy). Separation was performed on a Kinetex RP C18 column, 5 μm particle size, 50 × 2.0 mm (Phenomenex, Torrance, CA, USA). The mobile phase was (A) water and (B) acetonitrile, both containing 0.1% (v/v) acetic acid. The mobile phase gradient was from 90% to 10% A in 10 min (0.2 mL/min flow rate). ESI conditions used in negative polarization mode for the full-scan method were as follows: needle potential –3500 V, shield –500 V and capillary –30 V. Gas conditions were set with 20.0 psi for nebulizing gas and 25.0 psi of N2 at 300°C as drying gas. The fractions containing pure isabelin were reunited, and the solvent was evaporated to dryness in order to obtain pure isabelin. The 1000 μg/mL stock solution was prepared by weighing isabelin and dissolving it in the corresponding amount of solvent (75% v/v acetonitrile or sterile demi water). Antibacterial and anti-yeast assays based on agar-diffusion test To test for antimicrobial activity against bacteria and the yeast, agar disk-diffusion tests were performed in 12-well plates (Balouiri, Sadiki and Ibnsouda 2016). Single colonies of E. coli WA321, S. aureus 533R4 and C. albicans BSMY 212 were picked from plates and grown overnight in liquid LB or YEPD media at 37°C, 220 rpm. Fresh LB agar or YEPD agar (1.0% Merck Agar) was prepared and cooled down to ∼45°C. The target organisms were added to a final OD600 of 0.002 corresponding to 6.0 × 105 CFU/mL (E. coli WA321), 4.0 × 105 CFU/mL (S. aureus 533R4) or 1.6 × 104 cells/mL (C. albicans BSMY 212). A volume of 1 mL of seeded agar was added to each well of the 12-well plates (Greiner bio-one, Cat# 665180). After solidification, a filter paper (diameter ∼5.5 mm) (Whatman, Cat# 1003–150, 6 μm pore size) was added on the top of the agar surface and a volume of 5 μl of 1000 μg/mL isabelin (=5 μg) was added to the filter paper. As control, 5 μl of the solvent (75% acetonitrile) was added. Additionally, for positive assay control, 5 μl of appropriate antibiotic (ampicillin 100 mg/mL for E. coli WA321, tetracycline 15 mg/mL for S. aureus 533R4 or cycloheximide 25 mg/mL for C. albicans BSMY 212) was used. As negative assay control, filter papers without added antibiotics or isabelin were applied. The plates were incubated overnight at 37°C and on the next day plates were examined for visible zones of inhibition (ZOI) around the filter papers. Digital photographs were taken and analyzed using the AXIO VISION v4.8 imaging Software (Carl Zeiss Imaging Solutions GmbH) for surface-area determination (in pixel2). All treatments were performed in quadruplicates. Bioassays to test for antifungal/anti-oomycetal activity based on agar-diffusion test To test the effect of isabelin on fungal and oomycetal growth (mycelial extension), the following fungi/oomycetes were used: Chaetomium sp., F. culmorum PV, F. oxysporum, F. solani, M. hiemalis, P. ultimum P17, R. solani AG2.2 IIIB, T. harzianum, V. albo-atrum. The assays were performed in 12-well plates (Greiner bio-one, Cat# 665180). For testing the effect of isabelin on mycelial growth, fresh PD agar (29 g L−1 Oxoid CM 139) was prepared and a volume of 1 mL PD agar was added to each well. After solidification, the target fungi/oomycete were added by placing a 5-mm diameter plug of each fungus/oomycete at the top edge of each well. A filter paper with a diameter of ∼5.5 mm (Whatman, Cat# 1003–150, 6 μm pore size) was placed on the agar surface at the lower edge of each compartment, and a volume of 5 μl stock solution (corresponding to 5 μg) isabelin was added to each filter paper. For control, 5 μl of the solvent (75% v/v acetonitrile) was used. Plates were incubated at 24°C for 4 days. After incubation, plates were examined for fungal growth (=mycelial extension) and digital photographs were taken. The digital images were analyzed using the AXIO VISION v4.8 imaging Software (Carl Zeiss Imaging Solutions GmbH) to compare the growth of the treatments to the controls without added isabelin. In vitro test of growth inhibition on soil bacteria in liquid media For the determination of antimicrobial activity of isabelin in liquid media optical density (absorbance), measurements and counting of colony forming units (CFU) were performed in 96-well plates (Greiner bio-one, Cat# 655180). For the in vitro test, six phylogenetically different soil bacteria were used: A. denitrificans sp. MH58.1, Ps. fluorescences sp. AD21, Se. plymuthica sp. PGPRI-1, St. maltophilia sp. MH58.27, X. campestris sp. V79 and Paenibacillus sp. AD87. Single colonies of each bacterial isolate were picked from plates and grown in 20 mL of one-tenth Tryptic Soy Broth (TSB) overnight at 24°C, 200 rpm. For the absorbance measurement, 20 μl of 1000 μg/mL isabelin dissolved in water (10% v/v) (=20 μg) was dispensed in 180 μl of one-tenth TSB containing the soil bacterial isolates diluted to OD600 of 0.05. As positive assay control, 5 μl of 15 mg/mL of tetracycline (=75 μg) (Sigma-Aldrich product T-7660) was applied. The growth rates were monitored in the presence of isabelin and were compared to the growth rates of the controls (soil bacteria in the absence of isabelin) using a BioTek Synergy HT Multi-Mode Microplate Reader (Beun de Ronde Life Sciences, The Netherlands). The absorbance was measured using the following settings: measure absorbance at 600 nm, 24 h of measurement with an interval of 1 h, delay between each sample measurement was set to 50 ms, shaking intensity was set to 1 and the duration to 15 min before each measurement. The entire assay was performed at 24°C for 24 h. All treatments were measured in three replicates. The measured values were normalized against the mean absorbance values of the used culture media. In vitro test of growth inhibition on human pathogenic bacteria and yeast model organism The effect of pure isabelin was tested on three human pathogenic model organisms. For this, optical density measurements and counting of CFU were performed. Single colonies of each bacterial target organism, E. coli WA321, S. aureus 533R4 and of the yeast like fungi C. albicans BSMY 212, were picked from plates and grown overnight in 20 mL of LB broth or YEPD broth at 37°C, 220 rpm. Fresh LB or YEPD growth media was supplemented with the target organisms at a final OD600 of 0.002 corresponding to 6.0 × 105 CFU/mL (E. coli WA321), 4.0 × 105 CFU/mL (S. aureus 533R4) or 1.6 × 104 cells/mL (C. albicans BSMY 212). The assay was performed in 96-well plates (Greiner bio-one, Cat# 655180). For the measurement, 20 μl of 1000 ppm (equivalent to 20 μg) isabelin was dispensed in 180 μl of one-tenth LB or YEPD media containing the target organisms. As positive assay control, 5 μl of 15 mg/mL (=75 μg) of tetracycline (Sigma-Aldrich product T-7660) or 20 μl of 200 mg/mL (=4 mg) cycloheximide (Sigma-Aldrich) was applied. The growth rate of the model organisms was monitored in the presence of isabelin and was compared to the growth rates of the controls (in the absence of isabelin). The absorbance measurements were performed on a BioTek Synergy HT Multi-Mode Microplate Reader (Beun de Ronde Life Sciences, The Netherlands) with the following settings: measure absorbance at 600 nm, 24 h of measurement with 1-h interval, delay between each sample measurement 50 ms, shaking intensity 1 and 15 min between each measurement. The entire assay was performed at 37°C for 24 h. All treatments were measured in three replicates. The measured values were normalized against the mean absorbance values of the used culture media. Enumeration of target organism growth The growth of the target organisms grown in liquid was evaluated by plate counting. After 24 h of incubation and the optical density measurements, a sample of 100 μl of each well was taken and added to 900 μl of 10 mM phosphate buffer (KH2PO4, pH 6.5). Dilution series of each treatment were prepared in triplicates. A volume of 100 μl of each serial dilution was plated in three replicates with a disposable Drigalski spatula on one-tenth TSBA plates (soil bacteria), LB agar plates (human pathogenic model bacteria) or on YEPD agar plates (yeast model organism). The plates were incubated for 3 days at 24°C or overnight at 37°C, and CFU enumeration was carried out on an aCOlyte Colony Counter (Don Whitley Scientific, Meintrup DWS Laborgeräte GmbH, Germany). Statistical analysis Statistical analyses on fungal mycelial extension, the size of the ZOI and on viable count data (CFU) of bacteria and yeast-like fungi were performed with IBM SPSS Statistics 24 (IBM, Somers, NY, USA) using one-way ANOVA and post-hoc Tukey test (HSD) or two-tailed t-test between the data sets. Data were normalized by log transformation prior statistical analysis. The 5% level was taken as threshold for significance between the control and the treatments. RESULTS AND DISCUSSION Effect of isabelin on soil microorganisms Studying how plants and soil microorganisms interact is key to understand how plants can modify the soil environment by promoting or inhibiting specific soil microorganisms. Here we performed bioassays on commonly occurring soil microorganisms (fungi and bacteria) in order to determine potential growth inhibition or growth promotion by isabelin, a germacranolide sesquiterpene dilactone present in common ragweed extract, which has phytotoxic activity (Molinaro et al.2016). No changes in growth of fungi or oomycete could be observed in the presence of isabelin (Fig. 1). All tested soil fungi and the oomycete did not show significant differences of mycelial extension of the treatments compared to the controls. Figure 1. View largeDownload slide Effect of 5 μg isabelin dissolved in water (isabelin), water control (CTRL) on the growth of soil fungi and oomycetes. Effect on mycelial extension on (a) F. culmorum, (b) R. solani, (c) M. hiemalis and (d) P. ultimum. Data represent the mean of three replicates; error bars represent standard deviation. Figure 1. View largeDownload slide Effect of 5 μg isabelin dissolved in water (isabelin), water control (CTRL) on the growth of soil fungi and oomycetes. Effect on mycelial extension on (a) F. culmorum, (b) R. solani, (c) M. hiemalis and (d) P. ultimum. Data represent the mean of three replicates; error bars represent standard deviation. Although we did not observed any antifungal or anti-oomycetal activity exhibited by isabelin, which can be excreted by the invasive plant species A. artemisiifolia, other studies have demonstrated that invasive plant species are able to suppress arbuscular mycorrhizal fungi (Klironomos 2002; Callaway et al.2008; Rout and Callaway 2012). Bacterial enumeration of CFU after 24 h of growth in the presence of isabelin revealed the inhibition of only one of the tested soil bacteria, Paenibacillus sp. AD87 (Firmicutes) (Fig. 2). The growth of the Gram-positive Paenibacillus sp. AD87 was significantly inhibited (P < 0.001) by reaching 2.38 × 106 CFU/mL in presence of 20 μg isabelin compared to 2.64 × 107 CFU/mL in the control (Fig. 2). No effect by the presence of isabelin was observed on the other tested soil bacteria (A. denitrificans sp. MH58.1, Arthrobacter sp. V13, Burkholderia sp. AD24, Collimonas sp. Ter91, Ochrobactrum sp. 44, Ps. fluorescens sp. AD21, Se. plymuthica PGPRI-I, St. maltophilia sp. MH58.27, X. campestris sp. V79), belonging to the phyla of Actinobacteria, alpha-, beta- and gammaproteobacteria (Fig. S2, Table S1, Supporting Information). The genus Paenibacillus comprises bacterial species relevant to plants, rhizosphere and the soil environment. Many Paenibacillus species can promote crop growth directly via biological nitrogen fixation, phosphate solubilization, production of the phytohormone indole-3-acetic acid and the release of siderophores that enable iron acquisition. Furthermore, they can offer plant protection against insect herbivores and phytopathogens, including bacteria, fungi, nematodes and viruses (Ryu et al.2005; Anand, Grayston and Chanway 2013; Debois et al.2013). The observed specific effect of isabelin on the Paenibacillus genus may be providing a strategy of invasive plant to (i) inhibit this particular group of plant growth promoting bacteria (PGPR) and create an unfavorable environment for competitor plant species or (ii) to select specific microbiome for surrounding the roots of invasive plant species. Figure 2. View largeDownload slide CFUs of Paenibacillus sp. AD87 in the presence of 20 μg isabelin dissolved in water (isabelin), water control (CTRL) and 75 μg tetracycline as inhibition control (TC). Significant differences between treatments and the control are marked with an asterisk and the respective P-value. Data represent the mean of three replicates; error bars represent standard deviation. Figure 2. View largeDownload slide CFUs of Paenibacillus sp. AD87 in the presence of 20 μg isabelin dissolved in water (isabelin), water control (CTRL) and 75 μg tetracycline as inhibition control (TC). Significant differences between treatments and the control are marked with an asterisk and the respective P-value. Data represent the mean of three replicates; error bars represent standard deviation. Effect of isabelin on human pathogens Both the agar diffusion tests and liquid media bioassay on the human pathogens model organisms revealed inhibition on two of the three tested model species (Fig. 3). No inhibition was observed in the assays performed with E. coli. On the other hand, results obtained with S. aureus revealed a significant growth inhibition (P < 0.001) by reaching 1.33 × 106 CFU/mL in the presence of 20 μg isabelin compared to 8.44 × 108 CFU/mL in the control (Fig 3). Furthermore, the presence of 20 μg isabelin reduced absorbance values in OD600 measurement. This observation confirmed the appearance of the inhibition area (ZOI) in the agar-diffusion test. This clear difference in antimicrobial activity of isabelin observed against Gram-positive and Gram-negative bacteria is in line with other studies showing that Gram-positive bacteria are more sensitive to antimicrobial compounds than Gram-negative bacteria (Rice 2006; Giske et al.2008). Figure 3. View largeDownload slide Effect of 20 μg isabelin dissolved in water on the growth of three human pathogens: (a) E. coli WA321, (b) S. aureus 533R4, (c) C. albicans BSMY212. Controls: water control (CTRL) and 75 μg tetracycline (TC) or 4 mg Cycloheximide (CH) as inhibition control. Significant differences between treatments and the controls are marked with an asterisk and the respective P-value. Data represent the mean of three replicates; error bars represent standard deviation. Figure 3. View largeDownload slide Effect of 20 μg isabelin dissolved in water on the growth of three human pathogens: (a) E. coli WA321, (b) S. aureus 533R4, (c) C. albicans BSMY212. Controls: water control (CTRL) and 75 μg tetracycline (TC) or 4 mg Cycloheximide (CH) as inhibition control. Significant differences between treatments and the controls are marked with an asterisk and the respective P-value. Data represent the mean of three replicates; error bars represent standard deviation. The yeast-model organism, C. albicans, showed a significant (P = 0.001) decreased number of CFUs in the presence of 20 μg isabelin. Candida albicans reached 1.32 × 107 CFU/mL in the presence of 20 μg isabelin compared to 5.79 × 107 CFU/mL in the control (Fig. 3). This result is in accordance with the observed reduced absorbance values in the OD600 measurements. Candida species are ubiquitous common commensal yeasts, which are responsible for many nosocomial infections. Candida albicans, in particular, is responsible for more than half of all the candidemia cases (Lass-Flörl 2009). In the last decade, many Candida strains developed resistance to the azoles antibiotics, which are normally used for the treatment of these infections (Cleveland et al.2015). There is an increased interest in finding new active molecules with the ability of inhibiting Candida strains (Bona et al.2016). The cell wall organization in Gram-positive bacteria and Candida could possibly explain the observed growth-inhibitory effects of isabelin. Another sesquiterpene lactone, cnicin, a germacranolide sesquiterpene lactone found in Centauria species, is a molecule presenting Michael acceptors sites. Cnicin has been able to alkylate the thiol-groups of cysteine residue of the UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme involved in the peptidoglycan biosynthesis, causing the inhibition of the biosynthesis of the cell wall (Bachelier, Mayer and Klein 2006). The high reactivity of isabelin with thiol groups has been already demonstrated (Taglialatela-Scafati et al.2012), and the action on cell wall biosynthesis could be responsible for the antimicrobial activity of this compound. CONCLUSIONS This is the first study to test the antimicrobial activity of the pure sesquiterpene dilactone isabelin, produced by the invasive weed A. artemisiifolia, against a range of phylogenetically different soil-borne microorganisms and human pathogens. Isabelin did not reveal any antifungal or anti-oomycetal activity; however, it revealed strong antimicrobial activity against the Gram-positive soil bacteria Paenibacillus sp., the multidrug-resistant human pathogen S. aureus and the yeast human pathogen C. albicans. Many sesquiterpene lactones have been related to allergenic effects; however, little is known to date about the antimicrobial properties of these molecules. 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