Effects of Tripleurospermum caucasicum, Salvia rosmarinus and Tanacetum fruticulosum essential oils on aflatoxin B1 production and aflR gene expression in Aspergillus flavus.

Aflatoxin B1 (AFB1) is one of the most hazardous mycotoxins for humans and livestock that mainly produced by members of the genus Aspergillus in a variety of food commodities. In this study, the effect of S. rosmarinus, T. fruticulosum, and T. caucasicum essential oils (EOs) was studied on fungal growth, AFB1 production and aflR gene expression in toxigenic A. flavus IPI 247. The AFB1 producer A. flavus strain was cultured in YES medium in presence of various two-fold concentrations of the plant EOs (62.5-500 µg/mL) for 4 days at 28 °C. EO composition of plants was analyzed by Gas Chromatography/Mass Spectrometry (GC/MS). The amount of fungal growth, ergosterol content of fungal mycelia and AFB1 content of EO-treated and non-treated controls were measured. The expression of aflR gene was evaluated using Real-time PCR in the fungus exposed to minimum inhibitory concentration (MIC50) of EOs. The main constituents of the oils analyzed by GC/MS analysis were elemicin (33.80 %) and 2,3-dihydro farnesol (33.19 %) in T. caucasicum, 1,8-cineole (17.87 %), trans-caryophyllene (11.14 %), α and ẞ-pinene (10.92 and 8.83 %) in S. rosmarinus, and camphor (17.65 %), bornyl acetate (15.08 %), borneol (12.48 %) and camphene (11.72 %) in T. fruticulosum. The results showed that plant EOs at the concentration of 500 µg/mL suppressed significantly the fungal growth by 35.24-71.70 %, while mycelial ergosterol content and AFB1 production were inhibited meaningfully by 36.20-65.51 % and 20.61-89.16 %. T. caucasicum was the most effective plant, while T. fruticulosum showed the lowest effectiveness on fungal growth and AFB1 production. The expression of aflR in T. caucasicum and S. rosmarinus -treated fungus was significantly down-regulated by 2.85 and 2.12 folds, respectively, while it did not change in T. fruticulosum-treated A. flavus compared to non-treated controls. Our findings on the inhibitory activity of T. caucasicum and S. rosmarinus EOs toward A. flavus growth and AFB1 production could promise these plants as good candidates to control fungal contamination of agricultural crops and food commodities and subsequent contamination by AFB1. Down-regulation of aflR as the key regulatory gene in AF biosynthesis pathway warrants the use of these plants in AF control programs. Further studies to evaluate the inhibitory activity of studied plants EOs in food model systems are recommended.

Antimicrobial evaluation of Tripleurospermum callosum (Boiss. & Heldr.) E. Hossain extracts using in vitro and in vivo Caenorhabditis elegans model against urinary system pathogens.

Tripleurospermum callosum (Boiss. & Heldr.) E. Hossain was recorded in Turkish ethnobotanical data for its use against urinary and respiratory system ailments. Infusion, decoction and 96% ethanol extracts of T. callosum aerial parts were prepared for in vitro antimicrobial activity against urinary system pathogens Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Klebsiella aerogenes ATCC 1348 and Candida albicans ATCC 10231. The non-toxic concentrations of extracts and in vivo antimicrobial assay were performed using C. elegans. The extracts were analysed by Liquid Chromatography Mass Spectrometry (LC-MS/MS) for phytochemical composition. The water extracts were non-toxic at between 5000 and 312 µg/mL, while 96% ethanol extract at 312 µg/mL for C. elegans. The infusion extract showed in vivo anti-infective effect 5000-312 µg/mL against Gram-negative strains. The results indicate a potential role of plant extracts with relatively non-toxic and anti-infective effects against urinary system pathogens.

In vitro evaluation on the scolicidal effect of Myrtus communis L. and Tripleurospermum disciforme L. methanolic extracts.

Hydatid disease, a zoonotic disease, is still endemic in many developing countries that is caused by the metacestode of Echinococcus (E.) granulosus. Surgical management is one of the best choices for the treatment of the hydatidosis and using effective scolicidal agents during hydatid surgery is essential to prevent the secondary infection. The aim of the present in vitro study was to evaluate the scolicidal effect of the methanolic extract of Myrtus communis and Tripleurospermum disciforme against protoscoleces of hydatid cyst. Protoscoleces of E. granulosus were aspirated aseptically from infected livers. Various concentrations of M. communis and T. disciforme extracts at different exposure times were examined for their scolicidal activity. Normal saline and silver nitrate were used as negative and positive groups, correspondingly. The viability of protoscoleces was evaluated by 0.1% eosin. The result of the current study indicated that the highest scolicidal effect (100%) of M. communis was obtained at 100 and 50 mg/ml concentrations and LC50 in 10, 20 and 30 min were 11.64 mg/ml, 7.62 mg/ml, and 6.47 mg/ml respectively. The scolicidal activity of T. disciforme was negligible even at high concentration. The findings have shown that the scolicidal activity of M. communis against echinococcosis protoscoleces was strong, while the T. disciforme extract showed fewer effects. However, further studies are required for identification of the active ingredients in the extract and its safety on cells in effective concentrations.

A genome size and phylogenetic survey of Mediterranean Tripleurospermum and Matricaria (Anthemideae, Asteraceae).

The study of genome size variation can contribute valuable information on species relationships as well as correlate to several morphological or ecological features, among others. Here we provide an extensive report on genome sizes on genus Tripleurospermum and its closely related genus Matricaria, which are two typically Mediterranean genera particularly widespread and diverse in Turkey, the origin of most of the populations here studied. We analyse and discuss genome size variation in the first relatively complete molecular phylogenetic framework of Tripleurospermum (based on ITS and ETS ribosomal DNA-rDNA-regions). We find cases of intraspecific genome size variation, which could be taxonomically significant. Genome downsizing is also detected as the typical response to polyploidisation in Tripleurospermum taxa, being most conspicuous at the tetraploid level. Several positive correlations with genome size, including those with pollen and stomatal size or cypsela length, among others, are also found. Remarkably, taxa presenting rhizomes tend to present higher genome sizes, confirming a trend to accumulate nuclear DNA in such species, which could be explained by the nutrient reserves availability in their storage organs, allowing genome expansion, or by the lower rates of sexual reproduction in rhizomatous taxa.

Evaluation of chemical profile and antioxidant activity of Tripleurospermum insularum, a new species from Turkey.

This article presents the very first phytochemical investigation on new species Tripleurospermum insularum Inceer & Hayirlioglu-Ayaz. The volatile profile of odorous parts of the plant was analysed by GC/MS, and compounds were identified in headspace and essential oil obtained from aerial parts, representing 70.81% and 92.44% in total, respectively. The major volatiles were n-alkanes (38.43-59.22%), while essential oil was also rich in globulol (13.45%) and ß-sesquiphellandrene (9.29%). The content of phenolic compounds in methanolic extract and oil was 3621.62 and 14.4 mg GAE/100 g of dry plant, respectively. Moreover, potential medicinal effects were found in mean of antioxidant activity of this plant measured by using two different assays: radical-scavenging activity and ferric-reducing activity. Samples revealed values ranging from 0.33 to 146.80 µmol TE/100 g for DPPH assay, and from 2.29 to 5414.17 µmol AAE/100 g for FRAP assay.

Extraradical mycelium of arbuscular mycorrhizal fungi radiating from large plants depresses the growth of nearby seedlings in a nutrient deficient substrate.

The effect of arbuscular mycorrhiza (AM) on the interaction of large plants and seedlings in an early succession situation was investigated in a greenhouse experiment using compartmented rhizoboxes. Tripleurospermum inodorum, a highly mycorrhiza-responsive early coloniser of spoil banks, was cultivated either non-mycorrhizal or inoculated with AM fungi in the central compartment of the rhizoboxes. After two months, seedlings of T. inodorum or Sisymbrium loeselii, a non-host species colonising spoil banks simultaneously with T. inodorum, were planted in lateral compartments, which were colonised by the extraradical mycelium (ERM) of the pre-cultivated T. inodorum in the inoculated treatments. The experiment comprised the comparison of two AM fungal isolates and two substrates: spoil bank soil and a mixture of this soil with sand. As expected based on the low nutrient levels in the substrates, the pre-cultivated T. inodorum plants responded positively to mycorrhiza, the response being more pronounced in phosphorus uptake than in nitrogen uptake and growth. In contrast, the growth of the seedlings, both the host and the non-host species, was inhibited in the mycorrhizal treatments. Based on the phosphorus and nitrogen concentrations in the biomass of the experimental plants, this growth inhibition was attributed to nitrogen depletion in the lateral compartments by the ERM radiating from the central compartment. The results point to an important aspect of mycorrhizal effects on the coexistence of large plants and seedlings in nutrient deficient substrates.

Chemical composition and antibacterial activity of essential oils of Tripleurospermum disciforme in three developmental stages.

CONTEXT: Tripleurospermum disciforme (C.A. Mey) Schultz Bip. (Asteraceae) is a widespread biennial species which also has traditional medicinal uses. According to the few recent studies, essential oils of this species exhibit anti-inflammatory, antispasmodic, antiseptic, antifungal, antiulcer, and antioxidant activity. OBJECTIVE: The chemical compositions of the hydrodistilled oils of T. disciforme of Iranian origin are studied in the stages of prior to flowering, flowering, and post flowering, for the first time. Also, we investigated the antibacterial activities of the oils. MATERIALS AND METHODS: The essential oils of air-dried T. disciforme were obtained by hydrodistillation in three different developmental stages and analyzed by gas chromatography-mass spectrometry (GC-MS). The antimicrobial activity of the isolated essential oil, in the three stages, was also investigated against four Gram-positive and four Gram-negative bacteria. RESULTS: Twenty-one components were identified in the essential oils of T. disciforme, and the highest amount of oil was extracted at the flowering stage. The main component of the species in the flowering stage was ß-farnesene (22.46%) and the other major components were ß-sesquiphellandrene (17.85%), p-methoxy-ß-cyclopropylstyrene (16.64%), heptadecane (10.6%), p-methoxy-humulene oxide (6.88%) and benzene acetaldehyde (9.3%). The MIC of essential oil was evaluated from 4 µL ml(-1) against Staphylococcus subtilis and Bacillus cereus to 22 µL ml(-1) against Citrobacter amalonaticus. DISCUSSION AND CONCLUSION: This study demonstrates the occurrence of ß-farnesene/ß-sesquiphellandrene chemotype of T. disciforme in western regions of Iran that are different from previous reports. The findings also showed that the essential oils T. disciforme have excellent antibacterial activities and thus have great potentiality to be used as a resource for natural health products.

Development and activity of Glomus intraradices as affected by co-existence with Glomus claroideum in one root system.

The co-existence of two arbuscular mycorrhizal fungal (AMF) species, Glomus intraradices and Glomus claroideum, in the root systems of plants was investigated in a greenhouse experiment aimed at reconstructing interactions during an early stage of primary succession on a coal-mine spoil bank in Central Europe. Two plant species, Tripleurospermum inodorum and Calamagrostis epigejos, were inoculated either with one or both AMF species. Fungal development, determined by trypan blue and alkaline phosphatase staining as well as by PCR amplification of rRNA genes with species-specific primers, and the expression of five genes with different metabolic functions in the intraradical structures of G. intraradices were followed after 6 and 9 weeks of cultivation. The two AMF closely co-existed in the root systems of both plants possibly through similar colonisation rates and competitivity. Inoculation with the two fungi, however, did not bring any additional benefit to the host plants in comparison with single inoculation; moreover, plant growth depression observed after inoculation with G. claroideum persisted also in mixed inoculation. The expression of all the assayed G. intraradices genes was affected either by host plant or by co-inoculation with G. claroideum. The effects of both factors depended on the time of sampling, which underlines the importance of addressing this topic in time-course studies.

Combination effects of herbicides on plants and algae: do species and test systems matter?

Risk assessment of herbicides towards non-target plants in Europe is currently based solely on tests on algae and floating aquatic plants of Lemna sp. Effects on terrestrial non-target species is not systematically addressed. The purpose of the present study was to compare combination effects of herbicide mixtures across aquatic and terrestrial test systems, and to test whether results obtained in the traditional aquatic test systems can be extrapolated to the terrestrial environment. This was done by evaluating ten binary mixtures of nine herbicides representing the seven most commonly used molecular target sites for controlling broadleaved weeds. Data were evaluated statistically in relation to the concentration addition model, and for selected concentrations to the independent action model. The mixtures were tested on the terrestrial species Tripleurospermum inodorum (L.) Schultz-Bip. (Scentless Mayweed) and Stellaria media (L.) Vill. (Common Chickweed), and on the aquatic species Lemna minor L. (Lesser duckweed) and the alga Pseudokirchneriella subcapitata (Korschikov) Hindak. For the two mixtures of herbicides with the same molecular site of action, the joint effect was additive. For the eight mixtures of herbicides with different sites of action, two of the mixtures were consistently antagonistic across species, while for the remaining six mixtures the joint effect depended on the species tested. This dependence was, however, not systematic, in the sense that none of the species or test systems (terrestrial versus aquatic) had a significantly higher probability of showing synergistic or antagonistic joint effects than others. Synergistic interactions were not observed, but approximately 70% of the mixtures of herbicides with different sites of action showed significant antagonism. Hence, the concentration addition model can be used to estimate worst-case effects of mixtures of herbicides on both terrestrial and aquatic species. Comparing the sensitivity of the species to a 10% spray drift event showed that the terrestrial species were more vulnerable to all herbicides compared with the aquatic species, emphasising the importance of including terrestrial non-target plants in herbicide risk assessment.

Inhibition of carrageenan-induced edema by tripleurospermum disciforme extract in rats.

In this research anti-inflammatory effect of Tripleurospermum disciforme extract was studied in rats. The effect of the extract against acute inflammation was studied by hind paw edema test. Intraperitoneal injection of different doses (15, 30, 45, 60, 90 and 120 mg kg(-1)) of Tripleurospermum disciforme which was followed by hind paw carageenan injection 1 h later were investigated. The resultant edema was quantified by measuring changes of diameter of hind foot. After 2, 3, 4 and 6 h we measured the anti-inflammatory effect of Tripleurospermum disciforme extract. Tripleurospermum disciforme extract with doses of 45, 60, 90 and 120 mg kg(-1) and indomethacin 5 mg kg(-1) showed significant effect. It is concluded that Tripleurospermum disciforme extract has anti-inflammatory effect against acute inflammation.