Maurizio Sajeva

Molecular Characterization of Fusarium oxysporum f. melongenae by ISSR and RAPD Markers on Eggplant

Fusarium oxysporum f. melongenae is a major soil-borne pathogen of eggplant (Solanum melongena). ISSR and RAPD markers were used to characterize Fusarium oxysporum f. melongenae isolates collected from eggplant fields in southern Turkey. Those isolates were not pathogenic to tomato. Pathogens were identified by their morphology, and their identity was confirmed by PCR amplification using the specific primer PF02-3. The isolates were classified into groups on the basis of ISSR and RAPD fingerprints, which showed a level of genetic specificity and diversity not previously identified in Fusarium oxysporum f. melongenae, suggesting that genetic differences are related to the pathogen in the Mediterranean region. The primers selected to characterize Fusarium oxysporum f. melongenae may be used to determine genetic differences and pathogen virulence. This study is the first to characterize eggplant F.oxysporum species using ISSR and RAPD.

Essential oils composition of two Sicilian cultivars of Opuntia ficus-indica (L.) Mill. (Cactaceae) fruits (prickly pear)

The essential oils composition of the skin, pulp and seeds from fruits of two Sicilian cultivars of Opuntia ficus-indica (cv. Sanguigna and cv. Surfarina) has been obtained by hydrodistillation and the possible antioxidant, antimicrobial and semiochemical roles have been investigated comparing the data with those reported in the literature. The presence of antioxidants and antimicrobials found in this study increases the spectrum of compounds that have beneficial properties in O. ficus-indica. In addition, several compounds identified in this study have been reported to influence the behaviour of Ceratitis capitata, a phytophagous pest which causes severe damages to several crops including O. ficus-indica and the kairomonal activity of the odour of the fruits seems provided by a blend of compounds found in the various matrices analysed.

Dimethyl disulfide and dimethyl trisulfide: so similar yet so different in evoking biological responses in saprophilous flies

Dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS) are used by saprophilous insects to locate breeding sites (decaying organic matter), and by brood-site deceptive flowers to attract such insects. However, little is known about the relative importance of these two compounds in eliciting electrophysiological and behavioral responses in the insects. Here, we compared the relative attractiveness of DMDS and DMTS to saprophilous flies in field choice experiments and tested whether potential differences in field responses can be explained by differences in electrophysiological antennal responses to these compounds. Field experiments revealed that the attractiveness of a mixture of these compounds is due to DMTS alone. This result was confirmed by electroantennographic recordings in which flies of four tested species of Calliphoridae (Lucilia sericata, L. caesar,Calliphora vicina,Protocalliphora azurea) and one Muscidae (Musca domestica) respond clearly to DMTS, but not to DMDS. In house flies, however, DMTS elicited electrophysiological responses only, not reflected in behavioral assays. Despite the fact that DMTS and DMDS exhibit similar chemical structures, both the electroantennographic and field responses from saprophilous flies to these two compounds strongly differed. Our study suggests that oligosulfide-responsive saprophilous flies rely on DMTS and not DMDS for finding appropriate breeding sites and that DMTS and not DMDS could act as a key mediator for pollinator attraction in brood-site deceptive plants.

Regulating Internet trade in CITES species.

International trade in species that are or may be endangered by collection from the wild is regulated under the Convention on International Trade in Endangered Species of wild fauna and flora (CITES) for 176 member States (Parties). Internet commerce is a relatively new route for such trade. In 2007, the CITES Secretariat asked Parties to collect information on internet wildlife trade and report problems and implemented regulations. The reports indicated it was difficult to even approximate the influence of e-commerce on CITES-listed species (CITES Secretariat 2009). We report a case study in which we quantified international transactions over an internet auction site of CITES-listed cacti and cross-checked them with CITES trade data. Our results were both surprising and alarming. Species protected under CITES are included in different CITES appendices according to their level of threat of extinction from international trade. Taxa listed in Appendix I are endangered and wild collection could cause their extinction, and taxa listed in Appendix II either may become endangered if wild collection is not regulated or are similar in appearance to other species listed in Appendix I or II (see http://www.cites.org for full definitions). The treaty operates through the issue and control of permits that apply to regular and internet trade. Trade in wild specimens is either prohibited (species listed in Appendix I) or regulated by permits (species listed in Appendix II), although some Parties implement stricter regulations. Export permits must also be issued for certified artificially propagated plants. International trade of plants listed in Appendix I is allowed only if they are artificially propagated. Plants listed in Appendix I that are artificially propagated for commercial purposes are included in Appendix II (Article VII, paragraph 4 of the Convention), and the exporting enterprises should be registered in accordance with Resolution of the Conference of the Parties 9.19 (Revised at CoP15). Parties must submit annual reports to the CITES Secretariat listing the number and type of permits and certificates granted, the States with which such trade occurred, and the quantities and types of specimens traded. Some Parties report actual trade, whereas others report the permits issued. Trade data from these reports are stored in the CITES Trade Database (CTD) (http://www.unep-wcmc-apps.org/citestrade/). We used this information to assess the implementation of the Convention for international trade in CITES species over the internet. We investigated e-commerce in 2010 for cacti as a case study. All members of the Cactaceae, with the exception of 3 genera (Pereskia, Pereskiopsis, and Quiabentia) are listed in either Appendix I or II. We also restricted our analyses to species listed in Appendix I because an export permit is mandatory to export and import live plants. We monitored buyer–seller interactions on an internet auction site (not identified here) and recorded sales of live plants that were successfully completed and for which we could identify the plants country of origin and its destination. We compared this information with data on export permits for live plants in the CTD. Although these report trade data, rather than individual permits, they should reflect the internet trade if export permits were applied for, as required for all such transactions involving CITES Parties (the case for all transactions we recorded). Trade within the European Union does not require CITES permits, so we excluded these transaction and sales to Parties that have submitted official reservations against being regulated by CITES for certain cacti. All relevant exporting Parties had submitted their 2010 reports by the time of our analyses. We monitored 24 sellers over 6 months, twice weekly, until 1000 cacti listed in Appendix I had been sold. There were 978 such sales of a single plant, 1 sale with 6 plants, and 1 sale with 16 plants. We checked all scientific names and controlled for nomenclature differences between the names used in auctions and the official names used on CITES permits. Subspecies were noted if considered valid by the CITES cacti checklist. It is possible that the CTD records corresponding to a particular transaction actually corresponded to a different transaction with the same details. Our figures therefore represent the maximum number of transactions for which CITES export permits could have been issued. We did not check whether species were traded under permits spanning multiple years or under invalid permits, but we expected these would have only minor effects. Our data set contained roughly a quarter of the cactus plants for which CITES permits were issued in 2010. There were large discrepancies in the number of plants for which permits were issued and the number of plants traded in online transactions (Table 1). Our results suggest that only 10% of the plants traded were even potentially legal. Major discrepancies were also apparent in the number of species and number of importing and exporting countries between the online auctions and permits issued for that year (Table 1). Table 1 Sales of cacti listed on CITES Appendix I on an internet auction site and permits issued by CITES Parties in 2010 We suspect that most transactions we recorded were of artificially propagated plants. The United States was the only Range State recorded as exporting native species (approximately 8% of the recorded transactions). Few of the cacti sold that were pictured on the website had visible characteristics that could plausibly be associated with the plant being of wild origin. We therefore expect the recorded transactions to have only minor effects, if any, on wild populations. Nonetheless, an export permit is mandatory to export and import these plants. Therefore, the potentially wide scale of the illegal global trade that our results suggest should raise concerns about the adequacy of the protection for CITES species. For example, wild populations of some cacti listed in Appendix I may number only a few dozen individuals in their natural habitat (Hernandez et al. 2010), for which collectors are willing to pay high prices (Robbins 2003). Internet auction sites should be monitored more widely to investigate trade in CITES species with the aim of more effectively regulating trade in rare plants.

First records of pollinators of two co-occurring Mediterranean Apocynaceae

Abstract This article presents results of a field survey of pollinators of two Apocynaceae, Periploca laevigata subsp. angustifolia (Labill.) Markgraf (Periplocoideae) and Caralluma europaea (Guss.) N.E.Br. (Asclepiadoideae) co-occurring on Lampedusa Island, Mediterranean sea. Fifteen species within nine families of Diptera have been identified as pollinators of the two plants. The families involved are Tephritidae, Milichiidae, Trixoscelididae, Scathophagidae, Anthomyiidae, Muscidae, Calliphoridae, Sarcophagidae, Rhinophoridae. Families of Muscidae and Sarcophagidae are the more represented, respectively with four and three species. P. laevigata subsp. angustifolia seems to have a broader spectrum of pollinators, with 12 species of Diptera involved, while C. europaea has 8 species of Diptera as pollinators. Five species of Diptera are shared between the two plant species. The presented data are the first records of pollinators for P. laevigata subsp. angustifolia and C. europaea and confirm that both taxa are fly pollinated, though they do not conform exactly to the sapromyiophilous syndrome. The number of pollinators identified indicated that the morphological and functional floral specialization of the two taxa cover a wide number of pollinators with a similar biology.

Essential Oil Composition of Stems and Fruits of Caralluma europaea N.E.Br. (Apocynaceae)

The essential oil of the stems and fruits of Caralluma europaea (Guss.) N.E.Br. (Apocynaceae) from Lampedusa Island has been obtained by hydrodistillation and its composition analyzed. The analyses allowed the identification and quantification of 74 volatile compounds, of which 16 were aromatic and 58 non-aromatic. Stems and fruits contained 1.4% and 2.7% of aromatic compounds respectively, while non-aromatic were 88.3% and 88.8%. Non-aromatic hydrocarbons were the most abundant compounds in both organs, followed by fatty acids. Data showed differences in the profiles between stems and fruits which shared only eighteen compounds; stems accounted for 38 compounds while fruits for 53. Fruits showed a higher diversity especially in aromatic compounds with twelve versus four in stems. Among the volatiles identified in stems and fruits of C. europaea 26 are present in other taxa of Apocynaceae, 52 are semiochemicals for many insects, and 21 have antimicrobial activity. The possible ecological role of the volatiles found is briefly discussed.

Floral volatiles in a sapromyiophilous plant and their importance in attracting house fly pollinators

Floral scent in sapromyiophilous plants often consists of complex blends with not only fetid (e.g., sulfides) but also sweet (e.g., terpenoids) volatile organic compounds, and a recent study suggests that both groups of compounds are involved in pollinator attraction. However, little is known about the number and identity of compounds involved in pollinator attraction in these deceptive plants that mimic breeding sites of fly pollinators. In the present paper, we studied flower volatiles of sapromyiophilous Periploca laevigata and their capability to elicit biological responses in one of the pollinator species, Musca domestica. Floral volatiles were collected by dynamic headspace and analyzed by gas chromatography/mass spectrometry (GC/MS), and electrophysiological (GC/EAD) and behavioral assays (two choice olfactometer) were conducted. In the floral scent of P. laevigata, we detected 44 compounds, of which indole, β-caryophyllene, and germacrene D, as well as dimethyl trisulfide, which was present in trace amounts, were electrophysiologically active in the antennae of M. domestica. However, when we evaluated in behavioral experiments the attractiveness of the electrophysiologically active compounds (complete mixture against partial mixtures or against single compounds), we found that indole was the only attractive compound for the flies.

Headspace volatile composition of the flowers of Caralluma europaea N.E.Br. (Apocynaceae).

The volatile constituents of the flowers of Caralluma europaea (Guss.) N.E.Br (Apocynaceae) from Lampedusa Island were analyzed by a headspace GC method. The analyses allowed the identification and quantification of 41 compounds. The main components were, among the monoterpenoids, terpinolene (23.3%), α-terpinene (19.1%) and linalool (18.4%), whereas, among the carbonylic compounds the major constituents were heptanal (2.0%), octanoic acid (2.4%) and hexanoic acid (1.7%). The presence of a nitrogen containing compound, indole (0.8%) and of a sulphur containing compound, dimethylsulphide (t), noteworthy. The compounds found in the flowers of C. europaea have been compared with data available in the literature as regard to their odor, presence in other sapromyiophilous taxa, possible role as semiochemicals, and presence in decaying organic matter. 89.3% of total constituents have been described in other sapromyiophilous taxa. Some of the compounds are present in several types of decaying organic matter (excrements, decomposing bodies, and spoiled fish, etc). Several volatiles found in C. europaea flowers are used as semiochemicals by Hymenoptera, Coleoptera, Diptera, Lepidoptera and other insects. Sixteen volatiles, accounting for 32.4% of the total constituents, are described as attractants of some Diptera families, with a biology linked to decaying organic matter. Our data thus confirm that C. europaea floral bouquet falls within the sapromyiophilous pollination syndrome.

Essential oil composition of the fruits of Periploca laevigata Aiton subsp. angustifolia (Labill.) Markgraf (Apocynaceae – Periplocoideae)

The essential oil of the fruits of Periploca laevigata Aiton subsp. angustifolia (Labill.) Markgraf (Apocynaceae) from Lampedusa Island was obtained by hydrodistillation and its composition was analysed. The analyses allowed the identification and quantification of 64 volatile compounds belonging to different classes. The most abundant compounds were nonacosane, heptacosane, hentriacontane and δ-cadinene. Among the volatile compounds identified in the fruits of P. laevigata subsp. angustifolia, 31 are present in other taxa of Apocynaceae, 19 have antimicrobial activity and four are pheromones for the butterfly Danaus chrysippus. The possible ecological role of the volatile compounds found is briefly discussed.

Anti-tumor activity of the methanolic extracts of Salvia menthifolia

In the present research we investigated the anti-proliferative activity of Salvia menthifolia Ten. (formerly Salvia menthaefolia), Lamiaceae, on a glioblastoma cell line, since up to date poor therapeutic results have been reported for treatment of malignant glioblastoma. Methanol extracts from different anatomical parts of S. menthifolia were tested on DBTRG-05MG cell line by MTT assay. The most active primary stems extract was also evaluated for apoptosis induction. Results confirmed the anti-tumor property of all the organs and demonstrated that the primary stems extract induced apoptosis after 4 h with the highest values of DNA fragmentation after 6 to 24 h. Some extracts were also HPLC analyzed for polyphenols, althought activities could be due also to other constituents and to synergistic interactions. Rosmarinic acid, caffeic acid, luteolin-7-O-glucosyde and quercitrin were found in all the extracts. The good performance revealed for S. menthifolia towards this extremely aggressive human glioblastoma cell line confirms that the genus Salvia is a natural source of anti-tumor agents though there are great differences among the various species.