Genomic and proteomic analyses of the coral pathogen Vibrio coralliilyticus reveal a diverse virulence repertoire

Vibrio coralliilyticus has been implicated as an important pathogen of coral species worldwide. In this study, the nearly complete genome of Vibrio coralliilyticus strain P1 (LMG23696) was sequenced and proteases implicated in virulence of the strain were specifically investigated. The genome sequence of P1 (5 513 256 bp in size) consisted of 5222 coding sequences and 58 RNA genes (53 tRNAs and at least 5 rRNAs). Seventeen metalloprotease and effector (vgrG, hlyA and hcp) genes were identified in the genome and expressed proteases were also detected in the secretome of P1. As the VcpA zinc-metalloprotease has been considered an important virulence factor of V. coralliilyticus, a vcpA deletion mutant was constructed to evaluate the effect of this gene in animal pathogenesis. Both wild-type and mutant (ΔvcpA) strains exhibited similar virulence characteristics that resulted in high mortality in Artemia and Drosophila pathogenicity bioassays and strong photosystem II inactivation of the coral dinoflagellate endosymbiont (Symbiodinium). In contrast, the ΔvcpA mutant demonstrated higher hemolytic activity and secreted 18 proteins not secreted by the wild type. These proteins included four types of metalloproteases, a chitinase, a hemolysin-related protein RbmC, the Hcp protein and 12 hypothetical proteins. Overall, the results of this study indicate that V. coralliilyticus strain P1 has a diverse virulence repertoire that possibly enables this bacterium to be an efficient animal pathogen.     

New insights into Oculina patagonica coral diseases and their associated Vibrio spp. communities

Bleaching of Oculina patagonica has been extensively studied in the Eastern Mediterranean Sea, although no studies have been carried out in the Western basin. In 1996 Vibrio mediterranei was reported as the causative agent of bleaching in O. patagonica but it has not been related to bleached or healthy corals since 2003, suggesting that it was no longer involved in bleaching of O. patagonica. In an attempt to clarify the relationship between Vibrio spp., seawater temperature and coral diseases, as well as to investigate the putative differences between Eastern and Western Mediterranean basins, we have analysed the seasonal patterns of the culturable Vibrio spp. assemblages associated with healthy and diseased O. patagonica colonies. Two sampling points located in the Spanish Mediterranean coast were chosen for this study: Alicante Harbour and the Marine Reserve of Tabarca. A complex and dynamic assemblage of Vibrio spp. was present in O. patagonica along the whole year and under different environmental conditions and coral health status. While some Vibrio spp. were detected all year around in corals, the known pathogens V. mediteranei and V. coralliilyticus were only present in diseased specimens. The pathogenic potential of these bacteria was studied by experimental infection under laboratory conditions. Both vibrios caused diseased signs from 24 °C, being higher and faster at 28 °C. Unexpectedly, the co-inoculation of these two Vibrio species seemed to have a synergistic pathogenic effect over O. patagonica, as disease signs were readily observed at temperatures at which bleaching is not normally observed.     

Temperature-Regulated Bleaching and Lysis of the Coral Pocillopora damicornis by the Novel Pathogen Vibrio coralliilyticus

Coral bleaching is the disruption of symbioses between coral animals and their photosynthetic microalgal endosymbionts (zooxanthellae). It has been suggested that large-scale bleaching episodes are linked to global warming. The data presented here demonstrate that Vibrio coralliilyticus is an etiological agent of bleaching of the coral Pocillopora damicornis. This bacterium was present at high levels in bleached P. damicornis but absent from healthy corals. The bacterium was isolated in pure culture, characterized microbiologically, and shown to cause bleaching when it was inoculated onto healthy corals at 25°C. The pathogen was reisolated from the diseased tissues of the infected corals. The zooxanthella concentration in the bacterium-bleached corals was less than 12% of the zooxanthella concentration in healthy corals. When P. damicornis was infected with V. coralliilyticus at higher temperatures (27 and 29°C), the corals lysed within 2 weeks, indicating that the seawater temperature is a critical environmental parameter in determining the outcome of infection. A large increase in the level of the extracellular protease activity of V. coralliilyticus occurred at the same temperature range (24 to 28°C) as the transition from bleaching to lysis of the corals. We suggest that bleaching of P. damicornis results from an attack on the algae, whereas bacterium-induced lysis and death are promoted by bacterial extracellular proteases. The data presented here support the bacterial hypothesis of coral bleaching.     

Corals shed bacteria as a potential mechanism of resilience to organic matter enrichment

Understanding the mechanisms of resilience of coral reefs to anthropogenic stressors is a critical step toward mitigating their current global decline. Coral–bacteria associations are fundamental to reef health and disease, but direct observations of these interactions remain largely unexplored. Here, we use novel technology, high-speed laser scanning confocal microscopy on live coral (Pocillopora damicornis), to test the hypothesis that corals exert control over the abundance of their associated bacterial communities by releasing (‘shedding'') bacteria from their surface, and that this mechanism can counteract bacterial growth stimulated by organic inputs. We also test the hypothesis that the coral pathogen Vibrio coralliilyticus can evade such a defense mechanism. This first report of direct observation with high-speed confocal microscopy of living coral and its associated bacterial community revealed a layer (3.3–146.8 μm thick) on the coral surface where bacteria were concentrated. The results of two independent experiments showed that the bacterial abundance in this layer was not sensitive to enrichment (5 mg l⁻¹ peptone), and that coral fragments exposed to enrichment released significantly more bacteria from their surfaces than control corals (P<0.01; 35.9±1.4 × 10⁵ cells cm⁻² coral versus 1.3±0.5 × 10⁵ cells cm⁻² coral). Our results provide direct support to the hypothesis that shedding bacteria may be an important mechanism by which coral-associated bacterial abundances are regulated under organic matter stress. Additionally, the novel ability to watch this ecological behavior in real-time at the microscale opens an unexplored avenue for mechanistic studies of coral–microbe interactions.     

In vivo analgesic,antipyretic, and anti-inflammatory potential in Swiss albino mice and in vitro thrombolytic activity of hydroalcoholic extract from Litsea glutinosa leaves

Background

The study was conducted to evaluate the in vitro thrombolytic activity, and in vivo analgesic, anti-inflammatory and antipyretic potentials of different hydrocarbon soluble extracts of Litsea glutinosa leaves for the first time widely used in the folkloric treatments in Bangladesh. This work aimed to create new insights on the fundamental mechanisms of the plant extracts involved in these activities.

Results

In thrombolytic activity assay, a significant clot disruption was observed at dose of 1 mg/mL for each of the extracts (volume 100 μL) when compared to the standard drug streptokinase. The n-hexane, ethyl acetate, chloroform, and crude methanolic extracts showed 32.23 ± 0.26, 37.67 ± 1.31, 43.13 ± 0.85, and 46.78 ± 0.9% clot lysis, respectively, whereas the positive control streptokinase showed 93.35 ± 0.35% disruption at the dose of 30,000 I.U. In hot plate method, the highest pain inhibitory activity was found at a dose of 500 mg/kg of crude extract (15.54 ± 0.37 sec) which differed significantly (P <0.01 and P <0.001) with that of the standard drug ketorolac (16.38 ± 0.27 sec). In acetic acid induced writhing test, the crude methanolic extract showed significant (P <0.01 and P <0.001) analgesic potential at doses 250 and 500 mg/kg body weight (45.98 and 56.32% inhibition, respectively), where ketorolac showed 64.36% inhibition. In anti-inflammatory activity test, the crude methanolic extract showed significant (P <0.001) potential at doses 250 and 500 mg/kg body weight (1.51 ± 0.04 and 1.47 ± 0.03 mm paw edema, respectively), where ketorolac showed 1.64 ± 0.05 mm edema after 3 h of carrageenan injection. In antipyretic activity assay, the crude extract showed notable reduction in body temperature (32.78 ± 0.46°C) at dose of 500 mg/kg-body weight, when the standard (at dose 150 mg/kg-body weight) exerted 33.32 ± 0.67°C temperature after 3 h of administration.

Conclusions

Our results yield that the crude hydroalcoholic extract has better effects than the other in all trials. In the context, it can be said that the leaves of L. glutinosa possess remarkable pharmacological effects, and justify its traditional use as analgesic, antipyretic, anti-inflammatory, and thrombolytic agent.     

Effect of temperature and cycle length on microbial competition in PHB-producing sequencing batch reactor

The impact of temperature and cycle length on microbial competition between polyhydroxybutyrate (PHB)-producing populations enriched in feast-famine sequencing batch reactors (SBRs) was investigated at temperatures of 20 °C and 30 °C, and in a cycle length range of 1–18 h. In this study, the microbial community structure of the PHB-producing enrichments was found to be strongly dependent on temperature, but not on cycle length. Zoogloea and Plasticicumulans acidivorans dominated the SBRs operated at 20 °C and 30 °C, respectively. Both enrichments accumulated PHB more than 75% of cell dry weight. Short-term temperature change experiments revealed that P. acidivorans was more temperature sensitive as compared with Zoogloea. This is particularly true for the PHB degradation, resulting in incomplete PHB degradation in P. acidivorans at 20 °C. Incomplete PHB degradation limited biomass growth and allowed Zoogloea to outcompete P. acidivorans. The PHB content at the end of the feast phase correlated well with the cycle length at a constant solid retention time (SRT). These results suggest that to establish enrichment with the capacity to store a high fraction of PHB, the number of cycles per SRT should be minimized independent of the temperature.     

Concentration of antibodies against Porphyromonas gingivalis is increased before the onset of symptoms of rheumatoid arthritis

BackgroundThe periodontal pathogen Porphyromonas gingivalis is hypothesized to be important in rheumatoid arthritis (RA) aetiology by inducing production of anti-citrullinated protein antibodies (ACPA). We have shown that ACPA precede RA onset by years, and that anti-P. gingivalis antibody levels are elevated in RA patients. The aim of this study was to investigate whether anti-P. gingivalis antibodies pre-date symptom onset and ACPA production.MethodsA case–control study (251 cases, 198 controls) was performed within the Biobank of Northern Sweden. Cases had donated blood samples (n = 422) before the onset of RA symptoms by 5.2 (6.2) years (median (interquartile range)). Blood was also collected from 192 RA patients following diagnosis. Antibodies against P. gingivalis virulence factor arginine gingipainB (RgpB), and a citrullinated peptide (CPP3) derived from the P. gingivalis peptidylarginine deiminase enzyme, were analysed by ELISA.ResultsAnti-RgpB IgG levels were significantly increased in pre-symptomatic individuals (mean ± SEM; 152.7 ± 14.8 AU/ml) and in RA patients (114.4 ± 16.9 AU/ml), compared with controls (p < 0.001). Anti-CPP3 antibodies were detected in 5 % of pre-symptomatic individuals and in 8 % of RA patients, with elevated levels in both subsets (4.33 ± 0.59 and 9.29 ± 1.81 AU/ml, respectively) compared with controls (p < 0.001). Anti-CPP3 antibodies followed the ACPA response, with increasing concentrations over time, whilst anti-RgpB antibodies were elevated and stable in the pre-symptomatic individuals with a trend towards lower levels after RA diagnosis.ConclusionsAnti-P. gingivalis antibody concentrations were significantly increased in RA patients compared with controls, and were detectable years before onset of symptoms of RA, supporting an aetiological role for P. gingivalis in the development of RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-016-1100-4) contains supplementary material, which is available to authorized users.     

Antimicrobial Resistance of the Coral Pathogen Vibrio coralliilyticus and Caribbean Sister Phylotypes Isolated from a Diseased Octocoral

Vibrio coralliilyticus is a global marine pathogen that has been found to cause disease in several marine organisms, including corals. This study is the first report of the isolation of V. coralliilyticus from a diseased Caribbean octocoral, Pseudopterogorgia americana. Five sister phylotypes were positively identified using 16S rRNA gene sequencing, recA probes specific for V. coralliilyticus, and rep-PCR fingerprinting. The antimicrobial resistance was compared between pathogenic strains of V. coralliilyticus and the Caribbean strains. First, the antimicrobial resistance of V. coralliilyticus-type strain ATCC BAA-450 was determined using an agar-overlay antimicrobial bioassay at 24°C and 27°C, temperatures which are relevant to its known temperature-dependent virulence. From 108 distinct bacteria isolated from P. americana, 12 inhibited the V. coralliilyticus-type strain at 24°C and five at 27°C. Next, the phenotypic comparison of two Caribbean phylotypes and three V. coralliilyticus reference strains against a subset of 30 bacteria demonstrated a similar resistance trend. At both temperatures, the reference strains were inhibited by three bacteria isolates, while the Caribbean strains were inhibited by four to nine bacteria. Additionally, V. coralliilyticus-type strain ATCC BAA-450 and one of the Caribbean strains were inhibited by a higher number of bacteria at 24°C compared with 27°C. Together, these results highlight that V. coralliilyticus strains have antimicrobial resistance to the majority of coral-associated bacteria tested, which may be temperature-dependent in some strains. Furthermore, all V. coralliilyticus strains tested showed multi-drug resistance to a range of 11–16 (out of 26) commercial antibiotics. This study establishes V. coralliilyticus in association with a Caribbean octocoral and demonstrates its resistance to the antimicrobial activity of coral-associated bacteria and to commercial antibiotics.     

In vivo antipyretic,antiemetic, in vitro membrane stabilization,antimicrobial, and cytotoxic activities of different extracts from Spilanthes paniculata leaves

Background

The study was conducted to evaluate the in vitro antimicrobial activity, cytotoxic, and membrane stabilization activities, and in vivo antiemetic and antipyretic potentials of ethanolic extract, n-hexane and ethyl acetate soluble fractions of Spilanthes paniculata leaves for the first time widely used in the traditional treatments in Bangladesh.

Results

In antipyretic activity assay, a significant reduction (P < 0.05) was observed in the temperature in the mice tested. At dose 400 mg/kg-body weight, the n-hexane soluble fraction showed the effect (36.7 ± 0.63°C ) as like as the standard (dose 150 mg/kg-body weight) after 5 h of administration. Extracts showed significant (P < 0.001) potential when tested for the antiemetic activity compared to the standard, metoclopramide. At dose 50 mg/kg-body weight, the standard showed 67.23% inhibition, whereas n-hexane and ethyl acetate soluble fractions showed 37.53% and 24.93% inhibition of emesis respectively at dose 400 mg/kg-body weight. In antimicrobial activity assay, the n-hexane soluble fraction (400 μg/disc) showed salient activity against the tested organisms. It exerts highest activity against Salmonella typhi (16.9 mm zone of inhibition); besides, crude, and ethyl acetate extracts showed resistance to Bacillus cereus and Bacillus subtilis, and Vibrio cholera respectively. All the extracts were tested for lysis of the erythrocytes. At the concentration of 1mg/ml, ethanol extract, and n-hexane and ethyl acetate soluble fractions significantly inhibited hypotonic solution induced lysis of the human red blood cell (HRBC) (27.406 ± 3.57, 46.034 ± 3.251, and 30.72 ± 5.679% respectively); where standard drug acetylsalicylic acid (concentration 0.1 mg/ml) showed 77.276 ± 0.321% inhibition. In case of heat induced HRBC hemolysis, the plant extracts also showed significant activity (34.21 ± 4.72, 21.81 ± 3.08, and 27.62 ± 8.79% inhibition respectively). In the brine shrimp lethality bioassay, the n-hexane fraction showed potent (LC₅₀ value 48.978 μg/ml) activity, whereas ethyl acetate fraction showed mild (LC₅₀ value 216.77 μg/ml) cytotoxic activity.

Conclusions

Our results showed that the n-hexane extract has better effects than the other in all trials. In the context, it can be said that the leaves of S. paniculata possess remarkable pharmacological effects, and justify its folkloric use as antimicrobial, antipyretic, anti-inflammatory, and antiemetic agent. Therefore, further research may be suggested to find possible mode of action of the plant part.     

Effect of light conditions on anatomical and biochemical aspects of somatic and zygotic embryos of hybrid larch (Larix?×?marschlinsii)

Background and Aims In conifers, mature somatic embryos and zygotic embryos appear to resemble one another physiologically and morphologically. However, phenotypes of cloned conifer embryos can be strongly influenced by a number of in vitro factors and in some instances clonal variation can exceed that found in nature. This study examines whether zygotic embryos that develop within light-opaque cones differ from somatic embryos developing in dark/light conditions in vitro. Embryogenesis in larch is well understood both in situ and in vitro and thus provides a suitable system for addressing this question.Methods Features of somatic and zygotic embryos of hybrid larch, Larix × marschlinsii, were quantified, including cotyledon numbers, protein concentration and phenol chemistry. Somatic embryos were placed either in light or darkness for the entire maturation period. Embryos at different developmental stages were embedded and sectioned for histological analysis.Key Results Light, and to a lesser degree abscisic acid (ABA), influenced accumulation of protein and phenolic compounds in somatic and zygotic embryos. Dark-grown mature somatic embryos had more protein (91·77 ± 11·26 µg protein mg^–1 f.wt) than either dark-grown zygotic embryos (62·40 ± 5·58) or light-grown somatic embryos (58·15 ± 10·02). Zygotic embryos never accumulated phenolic compounds at any stage, whereas somatic embryos stored phenolic compounds in the embryonal root caps and suspensors. Light induced the production of quercetrin (261·13 ± 9·2 µg g^–1 d.wt) in somatic embryos. Mature zygotic embryos that were removed from seeds and placed on medium in light rapidly accumulated phenolics in the embryonal root cap and hypocotyl. Delaying germination with ABA delayed phenolic compound accumulation, restricting it to the embryonal root cap.Conclusions In larch embryos, light has a negative effect on protein accumulation, but a positive effect on phenol accumulation. Light did not affect morphogenesis, e.g. cotyledon number. Somatic embryos produced different amounts of phenolics, such as quercetrin, depending on light conditions. The greatest difference was seen in the embryonal root cap in all embryo types and conditions.     

Mortalities of Eastern and Pacific Oyster Larvae Caused by the Pathogens Vibrio coralliilyticus and Vibrio tubiashii

Vibrio tubiashii is reported to be a bacterial pathogen of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) and has been associated with major hatchery crashes, causing shortages in seed oysters for commercial shellfish producers. Another bacterium, Vibrio coralliilyticus, a well-known coral pathogen, has recently been shown to elicit mortality in fish and shellfish. Several strains of V. coralliilyticus, such as ATCC 19105 and Pacific isolates RE22 and RE98, were misidentified as V. tubiashii until recently. We compared the mortalities caused by two V. tubiashii and four V. coralliilyticus strains in Eastern and Pacific oyster larvae. The 50% lethal dose (LD₅₀) of V. coralliilyticus in Eastern oysters (defined here as the dose required to kill 50% of the population in 6 days) ranged from 1.1 × 10⁴ to 3.0 × 10⁴ CFU/ml seawater; strains RE98 and RE22 were the most virulent. This study shows that V. coralliilyticus causes mortality in Eastern oyster larvae. Results for Pacific oysters were similar, with LD₅₀s between 1.2 × 10⁴ and 4.0 × 10⁴ CFU/ml. Vibrio tubiashii ATCC 19106 and ATCC 19109 were highly infectious toward Eastern oyster larvae but were essentially nonpathogenic toward healthy Pacific oyster larvae at dosages of ≥1.1 × 10⁴ CFU/ml. These data, coupled with the fact that several isolates originally thought to be V. tubiashii are actually V. coralliilyticus, suggest that V. coralliilyticus has been a more significant pathogen for larval bivalve shellfish than V. tubiashii, particularly on the U.S. West Coast, contributing to substantial hatchery-associated morbidity and mortality in recent years.     

In vitro antioxidant capacity and free radical scavenging evaluation of active metabolite constituents of Newbouldia laevis ethanolic leaf extract

Background

The aim of the present study was to evaluate the in vitro antioxidant and free radical scavenging capacity of bioactive metabolites present in Newbouldia laevis leaf extract.

Results

Chromatographic and spectrophotometric methods were used in the study and modified where necessary in the study. Bioactivity of the extract was determined at 10 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml and 400 μg/ml concentrations expressed in % inhibition. The yield of the ethanolic leaf extract of N.laevis was 30.3 g (9.93%). Evaluation of bioactive metabolic constituents gave high levels of ascorbic acid (515.53 ± 12 IU/100 g [25.7 mg/100 g]), vitamin E (26.46 ± 1.08 IU/100 g), saponins (6.2 ± 0.10), alkaloids (2.20 ± 0.03), cardiac glycosides(1.48 ± 0.22), amino acids and steroids (8.01 ± 0.04) measured in mg/100 g dry weight; moderate levels of vitamin A (188.28 ± 6.19 IU/100 g), tannins (0.09 ± 0.30), terpenoids (3.42 ± 0.67); low level of flavonoids (1.01 ± 0.34 mg/100 g) and absence of cyanogenic glycosides, carboxylic acids and aldehydes/ketones. The extracts percentage inhibition of DPPH, hydroxyl radical (OH^.), superoxide anion (O₂^.-), iron chelating, nitric oxide radical (NO), peroxynitrite (ONOO⁻), singlet oxygen (¹O₂), hypochlorous acid (HOCl), lipid peroxidation (LPO) and FRAP showed a concentration-dependent antioxidant activity with no significant difference with the controls. Though, IC₅₀ of the extract showed significant difference only in singlet oxygen (¹O₂) and iron chelating activity when compared with the controls.

Conclusions

The extract is a potential source of antioxidants/free radical scavengers having important metabolites which maybe linked to its ethno-medicinal use.     

Anti-inflammatory components of the Vietnamese starfish Protoreaster nodosus

Background

In the present study, we examined the inhibitory effects of a methanolic extract, dichloromethane fraction, water layer, and polyhydroxylated sterols (1–4) isolated from the Vietnamese starfish Protoreaster nodosus on pro-inflammatory cytokine (IL-12 p40, IL-6, and TNF-α) production in LPS-stimulated bone marrow-derived dendritic cells (BMDCs) using enzyme-linked immunosorbent assays (ELISA).

Results

The methanolic extract and dichloromethane fraction exerted potent inhibitory effects on the production of all three pro-inflammatory cytokines, with IC₅₀ values ranging from 0.60 ± 0.01 to 26.19 ± 0.64 μg/mL. Four highly pure steroid derivatives (1–4) were isolated from the dichloromethane fraction and water layer of P. nodosus. Potent inhibitory activities were also observed for (25S) 5α-cholestane-3β,4β,6α,7α,8β,15α,16β,26-octol (3) on the production of IL-12 p40 and IL-6 (IC_50s = 3.11 ± 0.08 and 1.35 ± 0.03 μM), and for (25S) 5α-cholestane-3β,6α,8β,15α,16β,26-hexol (1) and (25S) 5α-cholestane-3β,6α,7α,8β,15α,16β,26-heptol (2) on the production of IL-12 p40 (IC_50s = 0.01 ± 0.00 and 1.02 ± 0.01 μM). Moreover, nodososide (4) exhibited moderate inhibitory effects on IL-12 p40 and IL-6 production.

Conclusion

This is the first report of the anti-inflammatory activity from the starfish P. nodosus. The main finding of this study is the identification oxygenated steroid derivatives from P. nodosus with potent anti-inflammatory activities that may be developed as therapeutic agents for inflammatory diseases.     

Temperature Effects on the Attachment of Pasteuria penetrans Endospores to Meloidogyne arenaria Race 1

Pasteuria penetrans is a gram positive bacterium that prevents Meloidogyne spp. from reproducing and diminishes their ability to penetrate roots. The attachment of the endospores to the cuticle of the nematodes is the first step in the life cycle of the bacterium and is essential for its reproduction. As a preliminary study to a field solarization test, the effects of temperature on the attachment of P. penetrans on Meloidogyne arenaria race 1 were investigated. Preexposing second-stage juveniles (J2) of M. arenaria to approximately 30 °C in water before exposing them to endospores increased their receptivity to endospore attachment when compared to treating J2 at 25 °C or 35 °C. In tests with soil, highest attachment occurred when J2 were incubated in soil infested with endospores and maintained at 20 °C to 30 °C for 4 days. Heating J2 in soil to sublethal temperatures (35 °C to 40 °C) decreased endospore attachment. Incubating P. penetrans endospores in soil at 30 °C to 70 °C for 5 hours a day over 10 days resulted in reductions of endospore attachment to nematodes as temperatures of incubation increased to 50 °C and higher.     

Essential oils from two Eucalyptus from Tunisia and their insecticidal action on Orgyia trigotephras (Lepidotera,Lymantriidae)

Background

Essential oils extracted from aromatic and medicinal plants have many biological properties and are therefore an alternative to the use of synthetic products. The chemical composition of essential oils from two medicinal plants (Eucalyptus globulus and E. lehmannii) was determined and, their insecticidal effects on the third and fourth larval stages of Orgyia trigotephras were assessed.

Results

Larvae were collected from Jebel Abderrahmane (North-East of Tunisia), conserved in groups of 50/box (21 × 10 × 10 cm) at a temperature of 25°C. Larvae were tested for larvicidal activities of essential oils. Each oil was diluted in ethanol (96%) to prepare 3 test solutions (S1 = 0.05%, S2 = 0.10% and S3 = 0.50%). Essential oils were used for contact, ingestion and Olfactory actions and compared to reference products (Bacillus thuringiensis and Decis). Olfactory action of essential oils shows that larvae mortality is higher than contact action, lower than ingestion action. MTM and FTM of S3 of E. lehmannii were respectively 1 h 32 min and 1 h 39 min are higher than those of E. globulus (MTM = 51 min and FTM = 1 h 22 min 34 sec). Contact action of E. lehmannii oil shows low insecticidal activity compared to E. globulus. MTM are respectively (1 min 52 sec and 1 min 7 sec), FTM are (2 min 38 sec, 1 min 39 sec), are the shortest recorded for S3, on the third stage of larvae. The fourth stage of larvae, MTM are (2 min 20 sec and 2 min 9 sec), FTM are (3 min 25 sec, 3 min 19 sec). Ingestion action of essential oils is longer than the contact action, since the time of death exceeds 60 minutes for all species.

Conclusion

Results shows that essential oils have a toxic action on nerves leading to a disruption of vital system of insects. High toxic properties make these plant-derived compounds suitable for incorporation in integrated pest management programs.     

Diversity and relationships of Crocus sativus and its relatives analysed by inter-retroelement amplified polymorphism (IRAP)

Background and Aims: Saffron (Crocus sativus) is a sterile triploid (2n = 3x = 24) cultivated species, of unknown origin from other diploid and polyploid species in the genus Crocus (Iridaceae). Species in the genus have high morphological diversity, with no clear phylogenetic patterns below the level of section Crocus series Crocus. Using DNA markers, this study aimed to examine the diversity and relationships within and between species of Crocus series Crocus.Methods: Eleven inter-retroelement amplified polymorphism (IRAP) primers were used in 63 different combinations with 35 single-plant accessions of C. sativus and related Crocus species in order to determine genetic variability and to conduct phylogenetic analysis.Key Results: A total of 4521 distinct polymorphic bands from 100 bp to approx. 4 kb were amplified; no fragment specific to all accessions of a single species was amplified. The polymorphic information content (PIC) values varied from approx. 0·37 to approx. 0·05 (mean 0·17 ± 0·1) and the major allele frequency had a mean of 0·87. High levels of polymorphism were identified between accessions of the six species of Crocus series Crocus related to C. sativus, with further variation between the species. In contrast, no polymorphisms were seen among 17 C. sativus accessions obtained in the region from Kashmir through Iran to Spain.Conclusions In contrast to the intraspecific variability seen in other Crocus species, C. sativus has minimal genetic variation, and it is concluded that the triploid hybrid species has most probably arisen only once. The data show that saffron is an allotriploid species, with the IRAP analysis indicating that the most likely ancestors are C. cartwrightianus and C. pallasii subsp. pallasii (or close relatives). The results may facilitate resynthesizing saffron with improved characteristics, and show the need for conservation and collection of wild Crocus.