Antimicrobial Activity of Scabiosa arenaria Forssk. Extracts and Pure Compounds Using Bioguided Fractionation

The emergence of multidrug resistant pathogens threatened the clinical efficacy of many existing antibiotics. This situation has been recognized globally as a serious concern and justifies further research to discover antimicrobial agents from natural origins including plant extracts. The aim of our work was to evaluate the antimicrobial activities of Scabiosa arenaria Forssk . extracts and pure compounds using a bioguided fractionation, and try to explain some traditional use of this genus. The best antimicrobial activity‐guided fractionation was obtained by BuOH fractions of flowers, fruits and (stems and leaves) against Escherichia coli, Pseudomonas aeruginosa and Candida albicans with minimum inhibitory concentration (MIC) values from 0.0195 to 5 mg/ml. Escherichia coli was the most affected bug, thus the MIC of fruits BuOH extract showed the best anti‐Escherichia coli activity (MIC = 0.0195 mg/ml), followed by the (stems and leaves) and flowers BuOH extracts; MIC = 0.078 and 0.15 mg/ml, respectively. Furthermore, the subfractions obtained from these three mixed fractions showed also an important antimicrobial activity against the three microorganisms, with MIC values between 0.0195 and 0.312 mg/ml. The fractionation of the aerial part BuOH fraction led to the isolation of oleanolic acid ( 1 ) and luteolin 7‐O‐glucopyranoside ( 2 ) which are reported here for the first time from S. arenaria. Both compounds showed good antimicrobial activities with MIC values ranging from 170 to 683 μm and 86 to 347 μm , respectively. These results support the use of the Scabiosa genus to inhibit the growth of tested pathogenic bacteria and yeasts which may reduce illnesses associated with their exposure.     

Antimicrobial and Cytotoxic Activity of Extracts of Ferula heuffelii Griseb. ex Heuff. and Its Metabolites

The antimicrobial and cytotoxic activities of isolates (CHCl₃ and MeOH extracts and selected metabolites) obtained from the underground parts of the Balkan endemic plant Ferula heuffelii Griseb . ex Heuff . were assessed. The CHCl₃ and MeOH extracts exhibited moderate antimicrobial activity, being more pronounced against Gram‐positive than Gram‐negative bacteria, especially against Staphylococcus aureus (MIC=12.5 μg/ml for both extracts) and Micrococcus luteus (MIC=50 and 12.5 μg/ml, resp.). Among the tested metabolites, (6E)‐1‐(2,4‐dihydroxyphenyl)‐3,7,11‐trimethyl‐3‐vinyldodeca‐6,10‐dien‐1‐one ( 2 ) and (2S*,3R*)‐2‐[(3E)‐4,8‐dimethylnona‐3,7‐dien‐1‐yl]‐2,3‐dihydro‐7‐hydroxy‐2,3‐dimethylfuro[3,2‐c]coumarin ( 4 ) demonstrated the best antimicrobial activity. Compounds 2 and 4 both strongly inhibited the growth of M. luteus (MIC=11.2 and 5.2 μM , resp.) and Staphylococcus epidermidis (MIC=22.5 and 10.5 μM , resp.) and compound 2 additionally also the growth of Bacillus subtilis (MIC=11.2 μM ). The cytotoxic activity of the isolates was tested against three human cancer cell lines, viz., cervical adenocarcinoma (HeLa), chronic myelogenous leukemia (K562), and breast cancer (MCF‐7) cells. The CHCl₃ extract exhibited strong cytotoxic activity against all cell lines (IC₅₀<11.0 μg/ml). All compounds strongly inhibited the growth of the K562 and HeLa cell lines. Compound 4 exhibited also a strong activity against the MCF‐7 cell line, comparable to that of cisplatin (IC₅₀=22.32±1.32 vs. 18.67±0.75μM ).     

Antimicrobial activities of extracts of Cyperus rotundus L. rhizomes against some bacterial and fungal pathogens of strawberry and tomato

Antimicrobial activities of rhizome extracts of Cyperus rotundus were investigated on selected plant pathogenic bacteria and fungi. Ethyl acetate and hexane extracts showed antibacterial activity against three isolates of Clavibacter michiganensis subsp. michiganensis at concentrations of 900 and 1000 μg/ml. However, Gram-negative bacterial pathogens of tomato; Pseudomonas syringae pv. tomato and Ralstonia solanacearum were not inhibited from the extracts. Ethyl acetate extracts at 100 μg/ml inhibited mycelial growth and spore germination of the two strawberry isolates of Botrytis cinerea; however, no significant inhibition was found in tomato fungal pathogen, Fusarium oxysporum f. sp. radicis lycopersici. Minimum inhibitory concentrations were determined as 0.0625 and 0.125 mg/ml against C. michiganensis subsp. michiganensis for ethyl acetate and hexane extracts of rhizomes, respectively. This study shows the potentials of extracts of C. rotundus rhizomes as antimicrobial agents that are effective against the tested plant pathogenic bacteria and fungi.     

Antimicrobial activity of fatty acids from fruits of Peucedanum cervaria and P. alsaticum

Plants of the genus Peucedanum have been used in traditional medicine for a long time to treat different diseases including infectious diseases. The hexane fruits extracts of Peucedanum cervaria and P. alsaticum were examined for antimicrobial activity and analyzed for their fatty acid content. Fatty acid composition of oils were analyzed by GC/FID in methyl ester form. Minimal inhibitory concentrations (MICs) of fatty acid fractions against twelve reference bacterial and yeast strains were performed by the twofold serial microdilution broth method. Fourteen fatty acids were identified. Oleic and linoleic acids were found to be dominant. The extracts from both plants examined exhibited inhibitory effects against Gram‐positive strains tested with different MIC values (0.25–2 mg/ml); however, extract from P. alsaticum possessed stronger antibacterial properties and a broader spectrum. The growth of Gram‐negative bacteria and Candida spp. strains was not inhibited even at the highest extract concentration used (MIC>4 mg/ml). Standard fatty acids exhibited inhibitory effects towards all bacterial and yeast strains used in this study; however, the majority of bacteria were more sensitive to linoleic than to oleic acid. These results revealed, for the first time, that hexane extracts obtained from fruits of P. alsaticum and P. cervaria possess moderate in vitro antibacterial activity against Gram‐positive bacteria including staphylococci. Linoleic and oleic acids appear to be the compounds responsible for this effect, and a synergistic antimicrobial effect between these two fatty acids was indicated.     

A bacterial type III effector targets plant vesicle-associated membrane proteins

The soilborne bacterial pathogen Ralstonia solanacearum is one of the most destructive plant pathogens worldwide, and its infection process involves the manipulation of numerous plant cellular functions. In this work, we found that the R. solanacearum effector protein RipD partially suppressed different levels of plant immunity triggered by R. solanacearum elicitors, including specific responses triggered by pathogen-associated molecular patterns and secreted effectors. RipD localized in different subcellular compartments in plant cells, including vesicles, and its vesicular localization was enriched in cells undergoing R. solanacearum infection, suggesting that this specific localization may be particularly relevant during infection. Among RipD-interacting proteins, we identified plant vesicle-associated membrane proteins (VAMPs). We also found that overexpression of Arabidopsis thaliana VAMP721 and VAMP722 in Nicotiana benthamiana leaves promoted resistance to R. solanacearum, and this was abolished by the simultaneous expression of RipD, suggesting that RipD targets VAMPs to contribute to R. solanacearum virulence. Among proteins secreted in VAMP721/722-containing vesicles, CCOAOMT1 is an enzyme required for lignin biosynthesis, and mutation of CCOAOMT1 enhanced plant susceptibility to R. solanacearum. Altogether our results reveal the contribution of VAMPs to plant resistance against R. solanacearum and their targeting by a bacterial effector as a pathogen virulence strategy.     

Conidiogenones H and I, two new diterpenes of Cyclopiane class from a marine-derived endophytic fungus Penicillium chrysogenum QEN-24S

Two new tetracyclic diterpenes of the rarely reported cyclopiane class, conidiogenones H and I ( 1 and 2 , resp.), along with five related congeners, conidiogenones B – D and F ( 3 – 5 and 6 , resp.) and conidiogenol ( 7 ), were characterized from the culture extracts of Penicillium chrysogenum QEN‐24S, an endophytic fungus derived from an unidentified marine red algal species of the genus Laurencia. The structures of these compounds were established on the basis of extensive spectroscopic analysis. The inhibitory activity of theses diterpenes against four bacteria and one pathogen fungus was evaluated. Conidiogenone B ( 3 ) showed potent activity against Methicillin resistant Staphylococcus aureus (MRSA), Pseudomonas fluorescens, P. aeruginosa, and Staphylococcus epidermidis (each with a MIC value of 8 μg/ml), while conidiogenol ( 7 ) showed obvious activity against P. fluorescens and S. epidermidis (each with a MIC value of 16 μg/ml). This is the first report on antimicrobial activity of cyclopiane diterpenes.     

Interactions of Ralstonia solanacearum and Pectobacterium carotovorum with Meloidogyne incognita on potato

Effect of interactions of Meloidogyne incognita with Ralstonia solanacearum and interaction of M. incognita with Pectobacterium carotovorum were studied in sequential and simultaneous inoculations on potato (Solanum tuberosum). Inoculation of M. incognita caused a lesser reduction in plant growth than caused by R. solanacearum. Inoculation of M. incognita plus R. solanacearum caused a greater reduction in plant growth than the damage caused by either pathogen. Inoculation of M. incognita prior to R. solanacearum resulted in a greater reduction in plant growth than R. solanacearum was inoculated prior to M. incognita. However, inoculation of M. incognita or P. carotovorum caused similar reduction in plant growth. Inoculation of P. carotovorum prior to M. incognita caused lesser reduction in plant growth than simultaneous inoculation of both pathogens. Inoculation of M. incognita caused galling in potato roots but the size of galls was small. Inoculation of P. carotovorum or R. solanacearum with M. incognita had adverse effect on galling and nematode multiplication. Wilting or soft rot index was 3 when R. solanacearum or P. carotovorum was inoculated alone. In other treatments, where R. solanacearum or P. carotovorum was inoculated with M. incognita, wilting or soft rot indices were 5.     

The population genetic test Tajima's D identifies genes encoding pathogen‐associated molecular patterns and other virulence‐related genes in Ralstonia solanacearum

The detection of pathogen‐associated molecular patterns (PAMPs) by plant pattern recognition receptors (PRRs) is an essential part of plant immunity. Until recently, elf18, an epitope of elongation factor‐Tu (EF‐Tu), was the sole confirmed PAMP of Ralstonia solanacearum, the causal agent of bacterial wilt disease, limiting our understanding of R. solanacearum–plant interactions. Therefore, we set out to identify additional R. solanacearum PAMPs based on the hypothesis that genes encoding PAMPs are under selection to avoid recognition by plant PRRs. We calculated Tajima's D, a population genetic test statistic which identifies genes that do not evolve neutrally, for 3003 genes conserved in 37 R. solanacearum genomes. The screen flagged 49 non‐neutrally evolving genes, including not only EF‐Tu but also the gene for Cold Shock Protein C, which encodes the PAMP csp22. Importantly, an R. solanacearum allele of this PAMP was recently identified in a parallel independent study. Genes coding for efflux pumps, some with known roles in virulence, were also flagged by Tajima's D. We conclude that Tajima's D is a straightforward test to identify genes encoding PAMPs and other virulence‐related genes in plant pathogen genomes.     

Evaluation of the antimicrobial potential of two flavonoids isolated from limnophila plants

The antimicrobial potential of two bioflavonoids, i.e., 5,7‐dihydroxy‐4′,6,8‐trimethoxyflavone ( 1 ) and 5,6‐dihydroxy‐4′,7,8‐trimethoxyflavone ( 2 ), isolated from Limnophila heterophylla Benth . and L. indica (Linn .) Druce (Scrophulariaceae), respectively, were evaluated against the microbial strains Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Alternaria solani, and Candida albicans. Compounds 1 and 2 exhibited moderate but broad antimicrobial activities against both Gram‐positive and Gram‐negative bacteria and also against the fungal pathogens. Moreover, the mechanism of action of 1 and 2 on the cellular functions or structures of some of the microorganisms was studied. Compound 1 showed a bactericidal effect against E. coli and S. aureus (MICs of 200 and 250 μg/ml, resp.), while compound 2 was found to effectively kill B. subtilis by cell lysis. The growth of A. solani and C. albicans was inhibited by compounds 1 and 2 , respectively. The effects of the flavonoids on the cellular structures and the carbohydrate metabolic pathways were studied by scanning electron microscopy (SEM) of the treated cells and by assessing the specific activity of key enzymes of the pathways, respectively. At sublethal doses, they enhanced the activity of gluconeogenic fructose bisphosphatase, but decreased the activity of phosphofructokinase and isocitrate dehydrogenase, the key enzymes of the Embden Meyerhof Parnas pathway and the tricarboxylic acid cycle, respectively.     

Random Genome Sequencing of Ralstonia solanacearum Strain IVIA 1602 and Comparative Analysis with Strain GMI1000

Ralstonia solanacearum is a β‐proteobacterium which affects several hundred plant species and provokes important agronomic losses. Five biovars of this bacterium have been described and they show behavioural differences. In this study a random sequencing of the genome of R. solanacearum strain IVIA 1602 (race 3, biovar 2), isolated from potato, was performed. The resulting 730 Genomic Survey Sequences (GSSs), representing 6.38% of the complete genome, were compared against the completely sequenced genome of strain GMI1000 (race 1, biovar 3), isolated from tomato, which is the only strain of this species sequenced until now. This comparative analysis showed, as expected, a high degree of similarity, but it also revealed strain‐specific regions of the genome as well as a number of insertion/deletion events and chromosomal rearrangements. All together, this comparative analysis gives an overview of the genomic divergence between these two biovars of the R. solanacearum species complex.     

Chemical Composition and Antimicrobial Activity of Essential Oils from the Aerial Parts of Asteriscus graveolens (Forssk.) Less. and Pulicaria incisa (Lam.) DC.: Two Asteraceae Herbs Growing Wild in the Hoggar

In recent years, antimicrobial activities of essential oils have been intensively explored, mainly in researching and developing new antimicrobial agents to overcome microbial resistance. The present study investigates the chemical composition and antimicrobial activities of essential oils obtained from two Asteraceae: Asteriscus graveolens (Forssk .) Less . and Pulicaria incisa (Lam .) DC. Chemical analysis was performed using a combination of capillary GC‐FID and GC/MS analytical techniques. The major component of Asteriscus graveolens were cis‐chrysanthenyl acetate (31.1%), myrtenyl acetate (15.1%), and kessane (11.5%), while for Pulicaria incisa the main components were chrysanthenone (45.3%) and 2,6‐dimethylphenol (12.6%). The oils obtained from the aerial parts were tested against sixteen microbial strains by agar well diffusion technique and dilution methods and showed minimum inhibitory concentrations (MIC) in the range of 19 – 1250 μg/ml. A good antibacterial activity against a common nosocomial pathogen, Acinetobacter baumanniiATCC 19606 was observed, especially from Pulicaria incisa essential oil, with a MIC value up to 19 μg/ml. These results give significant information about the pharmacological activity of these essential oils, which suggest their benefits to human health, having the potential to be used for medical purposes.     

A New Antimicrobial Fatty Acid from the Calcareous Sponge Paragrantia cf. waguensis

A new acetylenic fatty acid, 1 , has been isolated from the title sponge. The structure of the molecule was elucidated to contain an enyne and a thiophene by spectroscopic methods. Compound 1 showed a weak cytotoxic effect against NBT‐T2 rat bladder epithelial cells (IC₅₀>20 μg/ml), and antimicrobial activity with minimal‐inhibitory concentrations (MIC) of 64 and 128 μg/ml against Staphylococcus aureus and Escherichia coli, respectively.     

Inhibitory effects of some invasive alien species leaf extracts against tomato (Lycopersicon esculentum Mill.) bacterial wilt (Ralstonia solanacearum)

The inhibitory activity of selected invasive alien species leaf extracts against tomato bacterial wilt was studied in vitro and greenhouse. In vitro inhibitory activity was undertaken in a disc diffusion sensitivity test. Furthermore, aqueous extracts of Eichhorina crassipes, Mimosa diplotricha and Lantana camara and methanolic extract of Prosopis juliflora which showed better inhibitory effect in vitro were evaluated against R. solanacearum in greenhouse on tomato. The plant extracts were applied and evaluated at three different times of application (at the time of inoculation; two days before pathogen inoculation; and two days after pathogen inoculation). The result showed that most of the treatment combinations significantly reduce disease incidence and area under disease progress curve and increase biomass of tomato plants, but the effectiveness of tested plant species depends on the type of plant species and application time. The application of plant extracts at the time of pathogen inoculation resulted in highest reduction of disease development on tomato plants. Aqueous extract of E. crassipes was found to be the most effective plant extract in disease suppression and increase above-ground biomass compared to inoculated control. The study revealed that the tested plant species have a potential of inhibiting the development of bacterial wilt on tomato.     

Essential Oils and Diethyl Ether Extracts of Serbian Xeranthemum cylindraceum and X. annum: Chemical Composition,Antimicrobial Activity,and Chemotaxonomic Implications

Detailed GC and GC‐MS analyses of the essential oils and Et₂O extracts of two Xeranthemum species – X. cylindraceum and X. annum – resulted in the identification of 254 components, in total. Terpenoids constituted the major part of both X. cylindraceum and X. annum essential oils and extracts (51.8–65.7%, and 50.7%, resp.). Among the sesquiterpenoids, the extracts of both investigated taxa contained the guaianolide xerantholide, its 11,13‐dihydro derivatives, and two additional sesquiterpene lactones: an eudesmanolide, 11,13‐dihydroisoalantolactone, and a pseudoguaianolide, confertin. The last two lactones and both isomers of 11,13‐dihydroxerantholide have not been previously detected in Xeranthemum species. The isolated extracts of X. cylindraceum and X. annum were tested in a broth microdilution assay against a panel of microorganisms. The tested extracts demonstrated significant antimicrobial inhibitory activity, ranging from 30 to 260 μg/ml, being most active against Bacillus cereus and Staphylococcus aureus, an important human pathogen, with MIC close in value to those of chloramphenicol. Chemotaxonomic significance of the sesquiterpene lactones' distribution in the taxa investigated in this study and those detected earlier in phylogenetically close species (up to the level of the tribe Cardueae) was also discussed.     

The Relation of Different Crop Roots Exudates to the Survival and Suppressive Effect of Stenotrophomonas maltophilia (PD4560), Biocontrol Agent of Bacterial Wilt of Potato

Experiments were performed under greenhouse conditions to control bacterial wilt of potato (potato brown rot), caused by Ralstonia solanacearum race 3 biovar 2, Phylotype II, sequevar 1 using various biocontrol strategies. These strategies involved the use of the bacterial biocontrol agent Stenotrophomonas maltophilia (PD4560), in clay or sandy soils, planted with cowpea, maize or tomato which was grown separately in different pots in the inoculated soils. After harvest, the soil derived from each cultivated crop was inoculated with a mixture of three virulent R. solanacearum strains (K3, K10 and K16) to achieve a final concentration of 5 × 10⁸ cfu/g dry soil and used in pots under greenhouse conditions to cultivate potato seed tubers. The highest survival of S. maltophilia in soil (more than 160 days) coincided with a remarkable suppressing effect on disease incidence caused by R. solanacearum that expressed by wilt severity (up to 100% reduction), area under disease progress curve (AUDPC) (up to 99% reduction) and counts of the pathogen in soil (up to 75% reduction), rhizosphere (up to 80% reduction) and plant tissue (up to 97% reduction) of potato plants. The amino acid analysis of root exudates of crops under investigation revealed high percentages of asparagines (15.5–21%), glutamine (16–20%) and sulphur‐containing methionine (7–9%) in both of the cowpea and maize, respectively. In tomato root exudates, high percentages of arginine (around 26%) and lysine (around 23%) were detected. Methionine is known to favour the growth of S. maltophilia suggesting that especially cowpea and maize are suitable for crop rotation with potato and will enhance the sustainability of the biocontrol agent S. maltophilia.     

Evaluation of bacterial antagonists of Ralstonia solanacearum,causal agent of bacterial wilt of potato

Bacterial wilt of potato caused by Ralstonia solanacearum is one of the most destructive diseases in Kurdistan province, Iran. The objective of the present study was to evaluate antagonistic effects of some rhizobacteria isolated from the rhizosphere of potato plants against R. solanacearum, the agent of potato bacterial wilt. A total of 52 rhizobacteria were isolated and screened for in vitro antagonistic activity against R. solanacearum. Seven isolates with inhibiting effects of the pathogen were identified by phenotypic properties and partial sequencing of 16s rRNA as Pseudomonas fluorescens Pf11, P. fluorescens Pf16, Pseudomonas putida Pp17, Paenibacillus sp. Pb28 and Enterobacter sp. En38, Pseudomonas fluorescens Pp23 and Serratia sp. Se40. Strains Pf11, Pf16, Pp17 and Pb28 significantly inhibited the growth of the pathogen. Strains En38, Pp23 and Se40 showed a moderate or weak inhibition. During greenhouse study, strains were evaluated for their effects in reducing of disease and increasing biomass of potato plants. In according to greenhouse experiment results, isolates Pb28, Pp17 and Pf11significantly reduced disease by 55.56%, 51.50% and 38.58%, respectively. In addition, plant biomass significantly increased in plants treated with Pb28, Pp17, Pf11 and Pf16, compared to the control. Therefore, this study shows that these four strains have potential to be used as biocontrol agents against R. solanacearum. To confirm their effectiveness as commercial biocontrol agent, it is necessary to evaluate their efficiency in the field conditions in the next studies.     

Chemical Composition and Biological Activity of Centaurea baseri: New Species from Turkey

The genus Centaurea L. is one of the largest and important genera of Asteraceae family. Centaurea species have been widely used as herbal remedies in folk medicine for their antidandruff, antidiarrheic, antirheumatic, anti‐inflammatory, choleretic, diuretic, digestive, stomachic, astringent, antipyretic, cytotoxic, and antibacterial properties. Centaurea baseri Kose & Alan is a recently described local endemic species in Turkey and this is the first study on the chemical composition and bioactivity of its hydrodistilled essential oil and the crude extract. According to chromatospectral analysis, hexadecanoic acid (42.3%), nonacosane (8.2%), and heptacosane (8.0%) were the main compounds of the essential oil, while 16 compounds were determined in the MeOH extract using LC/MS. Furthermore, antimicrobial, antioxidant, and cytotoxic effects of the essential oil and the extract were evaluated in comparison with the standard agents. The extract showed strong antifungal effect against Candida utilis at the concentration of 60 μg/ml (MIC) where the EO showed growth inhibition at the concentration of 47.00 μg/ml (MIC) against pathogen Bacillus cereus. Both the essential oil and the extract did not show any selective antioxidant properties. The extract showed remarkably selective cytotoxic properties against MCF‐7, PANC‐1, A549, and C6 glioma cells.