Repellent activity of catmint, Nepeta cataria, and iridoid nepetalactone isomers against Afro-tropical mosquitoes, ixodid ticks and red poultry mites

The repellent activity of the essential oil of the catmint plant, Nepeta cataria (Lamiaceae), and the main iridoid compounds (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone, was assessed against (i) major Afro-tropical pathogen vector mosquitoes, i.e. the malaria mosquito, Anopheles gambiae s.s. and the Southern house mosquito, Culex quinquefasciatus, using a World Health Organisation (WHO)-approved topical application bioassay (ii) the brown ear tick, Rhipicephalus appendiculatus, using a climbing repellency assay, and (iii) the red poultry mite, Dermanyssus gallinae, using field trapping experiments. Gas chromatography (GC) and coupled GC-mass spectrometry (GC-MS) analysis of two N. cataria chemotypes (A and B) used in the repellency assays showed that (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone were present in different proportions, with one of the oils (from chemotype A) being dominated by the (4aS,7S,7aR) isomer (91.95% by GC), and the other oil (from chemotype B) containing the two (4aS,7S,7aR) and (4aS,7S,7aS) isomers in 16.98% and 69.83% (by GC), respectively. The sesquiterpene hydrocarbon (E)-(1R,9S)-caryophyllene was identified as the only other major component in the oils (8.05% and 13.19% by GC, respectively). Using the topical application bioassay, the oils showed high repellent activity (chemotype A RD₅₀ = 0.081 mg cm⁻² and chemotype B RD₅₀ = 0.091 mg cm⁻²) for An. gambiae comparable with the synthetic repellent DEET (RD₅₀ = 0.12 mg cm⁻²), whilst for Cx. quinquefasciatus, lower repellent activity was recorded (chemotype A RD₅₀ = 0.34 mg cm⁻² and chemotype B RD₅₀ = 0.074 mg cm⁻²). Further repellency testing against An. gambiae using the purified (4aS,7S,7aR) and (4aS,7S,7aS)-nepetalactone isomers revealed overall lower repellent activity, compared to the chemotype A and B oils. Testing of binary mixtures of the (4aS,7S,7aR) and (4aS,7S,7aS) isomers across a range of ratios, but all at the same overall dose (0.1 mg), revealed not only a synergistic effect between the two, but also a surprising ratio-dependent effect, with lower activity for the pure isomers and equivalent or near-equivalent mixtures, but higher activity for non-equivalent ratios. Furthermore, a binary mixture of (4aS,7S,7aR) and (4aS,7S,7aS) isomers, in a ratio equivalent to that found in chemotype B oil, was less repellent than the oil itself, when tested at two doses equivalent to 0.1 and 0.01 mg chemotype B oil. The three-component blend including (E)-(1R,9S)-caryophyllene at the level found in chemotype B oil had the same activity as chemotype B oil. In a tick climbing repellency assay using R. appendiculatus, the oils showed high repellent activity comparable with data for other repellent essential oils (chemotype A RD₅₀ = 0.005 mg and chemotype B RD₅₀ = 0.0012 mg). In field trapping assays with D. gallinae, addition of the chemotype A and B oils, and a combination of the two, to traps pre-conditioned with D. gallinae, all resulted in a significant reduction of D. gallinae trap capture. In summary, these data suggest that although the nepetalactone isomers have the potential to be used in human and livestock protection against major pathogen vectors, intact, i.e. unfractionated, Nepeta spp. oils offer potentially greater protection, due to the presence of both nepetalactone isomers and other components such as (E)-(1R,9S)-caryophyllene.     

Effects of Echinaforce treatment on ex vivo-stimulated blood cells

The herb Echinacea purpurea, also called purple coneflower, is regarded as an immune modulator. This study examined changes in cytokine production in blood samples from 30 volunteers before and during 8-day oral administration with an ethanolic extract of fresh Echinacea purpurea (Echinaforce^®). Daily blood samples were ex vivo stimulated by LPS/SEB or Zymosan and analysed for a series of cytokines and haematological and metabolic parameters. Treatment reduced the proinflammatory mediators TNF-α and IL-1β by up to 24% (p < 0.05) and increased anti-inflammatory IL-10 levels by 13% (p < 0.05) in comparison to baseline. This demonstrated a substantial overall anti-inflammatory effect of Echinaforce^® for the whole group (n = 28). Chemokines MCP-1 and IL-8 were upregulated by 15% in samples from subjects treated with Echinaforce^® (p < 0.05). An analysis of a subgroup of volunteers who showed low pre-treatment levels of the cytokines MCP-1, IL-8, IL-10 or IFN-γ (n = 8) showed significant stimulation of these factors upon Echinaforce^® treatment (30-49% increases; p < 0.05), whereas the levels in subjects with higher pre-treatment levels remained unaffected. We chose the term “adapted immune-modulation” to describe this observation. Volunteers who reported high stress levels (n = 7) and more than 2 colds per year experienced a significant transient increase in IFN-γ upon Echinaforce^® treatment (>50%). Subjects with low cortisol levels (n = 11) showed significant down-regulation of the acute-phase proteins IL1-β, IL-6, IL-12 and TNF-α by Echinaforce^® (range, 13-25%), while subjects with higher cortisol levels showed no such down-regulation. This is the first ex vivo study to demonstrate adapted immune-modulation by an Echinacea preparation. While Echinaforce^® did not affect leukocyte counts, we speculate that the underlying therapeutic mechanism is based on differential multi-level modulation of the responses of the different types of leukocytes. Echinaforce^® thus regulates the production of chemokines and cytokines according to current immune status, such as responsiveness to exogenous stimuli, susceptibility to viral infection and exposure to stress.     

Efficacy of an essentifal oil of Cinnamomum zeylanicum against Psoroptes cuniculi

The aim of the present study was to investigate the in vitro and in vivo acaricidal effects of an essential oil of Cinnamomum zeylanicun leaves on Psoroptes cuniculi, a mange mite. In vitro, 2.5 ml of the essential oil diluted at different concentrations, from 10% to 0.03%, in paraffin oil were added to Petri dishes containing all motile stages of P. cuniculi. Mites mortality observed in these dishes was compared with that observed in untreated and treated (AcaCerulen R) control plates. In vivo, one group of six P. cuniculi infected rabbits was topically treated two times at seven days interval with two ml of the essential oil at the concentration of 2.5% in paraffin oil and compared with untreated and treated (AcaCerulen R) control groups of six rabbits each. After 24 h of contact, all concentrations of essential oil between 0.10 and 10% showed a good in vitro acaricidal efficacy if compared with the untreated controls (p<0.01), but only the concentrations between 0.16 and 10% turned out as active as the drug. In vivo, the treatment with the essential oil cured all infested rabbits and no statistical differences were observed with the treated control group.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.     

Variability in response of four populations of Amblyseius largoensis (Acari: Phytoseiidae) to Raoiella indica (Acari: Tenuipalpidae) and Tetranychus gloveri (Acari: Tetranychidae) eggs and larvae

Raoiella indica (Acari: Tenuipalpidae) is a phytophagous mite that recently invaded the Neotropical region. A predatory mite Amblyseius largoensis (Acari: Phytoseiidae) has been found associated with R. indica in Florida. This study evaluated A. largoensis by determining its likelihood of consuming eggs and larvae of R. indica and Tetranychus gloveri (Acari: Tetranychidae) under no-choice and choice conditions. To detect variations in the response of A. largoensis to R. indica, four populations of predators were examined: (1) predators reared exclusively on R. indica in the laboratory for 2 years, (2) predators reared on T. gloveri in the laboratory for 2 months but reared on R. indica for 2 years previously, (3) predators collected from a field infested with R. indica, and (4) predators collected from a field that had never been infested with R. indica. Results of this study suggest that A. largoensis is likely to accept and consume high numbers of R. indica eggs regardless of their previous feeding experience. In contrast, all populations consumed relatively fewer R. indica larvae than the other prey tested. Predators previously exposed to R. indica were more likely to consume R. indica larvae. By contrast, predators not previously exposed to R. indica showed the lowest likelihood of choosing to feed on this prey item. Plasticity in the response of A. largoensis to R. indica larvae could be associated to selection, learning, or a combination of both. The possible implications of the observed differences in terms of biological control of R. indica are discussed.     

Application of vibrational spectroscopy in the quality assessment of Buchu oil obtained from two commercially important Agathosma species (Rutaceae)

Quality assessment of natural raw materials and derived consumer products is often done using conventional analytical techniques such as liquid and gas chromatography which are expensive and time consuming. This paper reports on the use of vibrational spectroscopy techniques as possible alternatives for the rapid and inexpensive assessment of the quality of ‘buchu oil’ obtained from two South African species; Agathosma betulina and Agathosma crenulata belonging to the Rutaceae family. Samples of A. betulina (55) and A. crenulata (16) were collected from different natural localities and cultivation sites in South Africa. The essential oil was obtained by hydrodistillation and scanned on Near infrared (NIR), mid infrared (MIR) and Raman spectrometers. The spectral data obtained was processed using chemometric techniques and orthogonal partial least squares discriminant analysis (OPLS-DA) was used to clearly differentiate between A. betulina and A. crenulata. The OPLS-DA technique also proved to be a useful tool to identify wave regions that contain biomarkers (peaks) that contributed to the separation of the two species. The three spectroscopy techniques were also evaluated for their ability to accurately predict the percentage composition of seven major compounds that occur in A. betulina ‘buchu’ oil. Using GC–MS reference data, calibration models were developed for the MIR, NIR and Raman spectral data to predict/profile the major compounds in ‘buchu oil’. A comparison of the three spectroscopy techniques showed that MIR together with PLS algorithms produced the best model (R²X = 0.96; R²Y = 0.88 and Q²Ycum = 0.85) for the quantification of six of the seven major oil constituents. The MIR model showed high predictive power for pseudo-diosphenol (R² = 0.97), isomenthone (R² = 0.97), menthone (R² = 0.90), limonene (R² = 0.91), pulegone (R² = 0.96) and diosphenol (R² = 0.85). These results illustrate the potential of MIR spectroscopy as a rapid and inexpensive alternative to predict the major compounds in buchu oil.     

A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [³⁵S]GTPγS functional assays were performed in the presence of putative δ₁-(DPDPE: agonist, BNTX: antagonist), δ₂-(agonist: deltorphin II, Ile^5,6-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ₂- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with K_D = 0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [³H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [³H]Ile^5,6-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49 ± 0.06 and 0.30 ± 0.01 nM potency against DPDPE and deltorphin II in the [³⁵S]GTPγS functional assay, respectively. The rank order of potency of putative δ₁- and δ₂-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ₁- and δ₂-opioid receptors could not be unequivocally distinguished in vitro.     

Virulence of Hypocreales fungi to pecan aphids (Hemiptera: Aphididae) in the laboratory

There is need for efficacious biocontrol agents for aphids in commercial orchards. As a preliminary step to this end we determined the virulence of several Hypocreales fungi to pecan aphids. In the first experiment we tested the virulence of Isaria fumosorosea (ARSEF 3581) blastospores to three pecan aphids Monellia caryella, Melanocallis caryaefoliae, and Monelliopsis pecanis under laboratory conditions. Rates of 1 × 10⁷ or 1 × 10⁸ spores per ml were applied in 2 ml via a spray tower to 90 mm Petri dishes containing 10 aphids each. Mortality and mycosis were determined after 24, 48 and 72 h. Treatment effects were observed by 48 h post-application, and by 72 h the higher application rate caused >90% mortality and mycosis in M. caryella and M. caryaefoliae, whereas <70% was observed in M. pecanis.We conducted two subsequent experiments (Experiments 2 and 3), using the same methodology, to compare the virulence of several Hypocreales species and strains against the aphid of primary economic concern to most pecan growers, M. caryaefoliae. In Experiment 2, we compared blastospores and conidia of two I. fumosorosea strains (ARSEF 3581 and ATCC 20874 [= strain 97]). The blastospores of ARSEF 3581 and conidia of ATCC 20874 showed higher virulence than other treatments and thus were included in Experiment 3, which also compared the virulence of conidia of Beauveria bassiana (GHA strain) and Metarhizium anisopliae (F52 strain). Results in Experiment 3 indicated the highest virulence in I. fumosorosea 3581 blastospores and M. anisopliae (F52) followed by I. fumosorosea (20874) conidia. The detection of pathogenicity to pecan aphids establishes the potential for commercial usage and additional study. Results reported here will narrow treatments to test in future greenhouse and field trials.     

Nematicidal activity of Haplophyllum tuberculatum and Plectranthus cylindraceus oils against Meloidogyne javanica

The potentials of Haplophyllum tuberculatum and Plectranthus cylindraceus oils to control Meloidogyne javanica were investigated in vitro and in a greenhouse. A mixture of Haplophyllum and Plectranthus oils (1:1) was highly toxic to M. javanica in vitro, as it killed all nematode juveniles and inhibited hatching of eggs at 12.5 μg/ml concentration after 24 h exposure time, as did carbofuran at the same concentration. In the green-house, tomatoes grown in soil treated with a combination (1:1) of the two oils developed fewer root galls than those grown in soil treated with higher doses of either oil. The oil mixture, at 2.5 and 5.0 μg/ml of soil, was not phytotoxic to tomato plants as evident from the appearance and height of plants after 12 weeks exposure time, compared to treatment over the same period at lower effective doses. The nematicidal activity of the combined essential oils was suggested by the presence of C₁₀ dienes, C₁₀ trienes and C₁₀ phenol.     

Efficacy of indigenous entomopathogenic nematodes (Rhabditida: Heterorhabditidae, Steinernematidae), from Rio Grande do Sul Brazil, against Anastrephafraterculus (Wied.) (Diptera: Tephritidae) in peach orchards

Laboratory, greenhouse, and field experiments were performed with the objective of selecting efficient indigenous strains of entomopathogenic nematodes (EPNs) from Rio Grande do Sul (RS) state, Brazil, for controlling the South American fruit fly, Anastrepha fraterculus (Wied.). Laboratory experiments were conducted in 24 well-plates filled with sterile sand and one insect per well. In greenhouse experiments, plastic trays filled with soil collected from the field were used, while in field experiments, holes were made in soil under the edge of peach tree canopies. Among 19 EPN strains tested, Heterorhabditis bacteriophora Poinar RS88 and Steinernema riobrave Cabanillas, Poinar, & Raulston RS59 resulted in higher A. fraterculus larval (pre-pupal) and pupal mortality, with LD₉₀ of 1630, 457 and 2851, 423 infective juveniles (IJs)/cm², respectively. Greenhouse experiments showed no differences in pupal mortality at 250 and 500 IJs/cm² of either nematode. In the field, H. bacteriophora RS88 and S. riobravae RS59 sprayed individually over natural and artificially infested fruit (250 IJs/cm²) resulted in A. fraterculus larval mortality of 51.3%, 28.1% and 20%, 24.3%, respectively. There was no significant difference in A. fraterculus pupal mortality sprayed with an aqueous suspension of either nematode; however, when using infected insect cadavers, H. bacteriophora RS88 was more efficient than S. riobrave RS59. Our results showed that H. bacteriophora RS88 was more virulent to insect larvae, with an efficient host search inside the infested fruit and control of pupae in the soil after being applied by aqueous suspension or infected cadavers.     

Characterization of an extracellular lipase from the biocontrol fungus, Nomuraea rileyi MJ, and its toxicity toward Spodoptera litura

An extracellular lipase from Nomuraea rileyi MJ was purified 23.9-fold with 1.69% yield by ammonium sulfate precipitation followed by Sephacryl S-100 HR column chromatography. By mass spectrometry and SDS-polyacrylamide gel electrophoresis, the molecular weight of the homogenous lipase was 81 kDa. The N-terminal sequence was determined as LeuSerValGluGlnThrLysLeuSerLysLeuAlaTyrAsnAsp and it showed no homology to sequences of known lipases. The optimum pH and temperature for activity were 8.0 and 35 °C, respectively. The enzyme was stable in the pH range 7.0-9.0 and at 15-35 °C for 1 h. Higher activity was observed in the presence of surfactants, Na⁺, NH₄⁺ ions, NaN₃ and ethylenediaminetetraacetic acid (EDTA), while Co²⁺ and Cu²⁺ ions, cysteine and dithiothreitol (DTT) strongly inhibited activity. The purified lipase hydrolyzed both synthetic and natural triglycerides with maximum activity for trilaurin and coconut oil, respectively. It also hydrolyzed esters of p-nitrophenol (pNP) with highest activity for p-nitrophenyl caprate (pNPCA). The purified lipase was found to promote N. rileyi spore germination in vitro in that germination reached 98% in conidial suspensions containing purified lipase at 2.75 U. Moreover, it enhanced toxicity of N. rileyi toward Spodoptera litura larvae with mortality via topical application reaching 63.3% at 4-10 days post-treatment which calculated to be 2.7 times higher than the mortality obtained using conidial suspensions alone.     

Lavandula angustifolia essential oil as a novel and promising natural candidate for tick (Rhipicephalus (Boophilus) annulatus) control

Lavandula angustifolia is a well known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of L. angustifolia essential oil (EO) against engorged Rhipicephalus (Boophilus) annulatus (Acari; Ixodidae) females. For this purpose six serial concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v) of L. angustifolia EO were used. There was considerable mortality in concentrations more than 4.0% although there were no differences between 6.0 and 8.0% in all measured criteria. The mortality rate 24 h after inoculation was 73.26 and 100% in groups treated with 4.0 and 8.0% EO, respectively. Lavender EO also reduced tick egg weight in a concentration-dependent manner. The amount of eggs produced varied from 0.12 g (at 0.5% EO) to 0.00 g (at 8.0% EO) but did not differ statistically from the control. L. angustifolia EO caused 100% failure in egg laying at 6.0 and 8.0% whereas this value in the control group was zero. A positive correlation between L. angustifolia EO concentration and tick control, assessed by relative mortality rate and egg-laying weight, was observed by the EO LC/EC₅₀, which, when calculated using the Probit test, was 2.76-fold higher than the control. Lavender is a promising acaricidal against R. (B.) annulatusin vitro.     

Microbial tolerance in secondary peritonitis is dose dependent

Local microbial tolerance was investigated in a murine model of peritonitis. Peritoneal bacterial burden and inflammatory cytokine concentrations were determined at different times, within 48 h after infection. Peritoneal macrophages were harvested from naïve mice or from mice 48 h after infection and underwent ex vivo stimulation with different concentrations of Klebsiella. Cytokine secretion was determined in the supernatants. Peritoneal bacteria concentrations, remained relatively steady between 24 h (median: 5.04 log CFU) and 48 h (median: 5.19 log CFU) after infection. Peritoneal cytokine concentrations peaked early but were already diminished at 48 h after infection, despite persistent high bacteria levels. Macrophages, harvested from naïve mice responded vigorously to ex vivo stimulation with 10⁵ CFU and 2 × 10⁸ CFU Klebsiella. Cells harvested from animals 48 h after infection, were unresponsive to an ex vivo stimulation with 10⁵ CFU Klebsiella, but fully responded to 10⁸ CFU. Persistent intraabdominal bacterial infection induced dose dependent microbial tolerance in peritoneal macrophages.     

Expression and characterization of the chitinases from Serratia marcescens GEI strain for the control of Varroa destructor, a honey bee parasite

Serratia marcescens GEI strain was isolated from the gut of the workers of Chinese honey bee Apis cerana and evaluated in the laboratory for the control of Varroa destructor, a parasite of western honey bee A. mellifera. The supernatant and the collected proteins by ammonium sulfate from the bacterial cultures showed a strong miticidal effect on the female mites, with 100% mite mortality in 5 days. Heat (100 °C for 10 min) and proteinase K treatment of the collected proteins destroyed the miticidal activity. The improved miticial activity of this bacterial strain on chitin medium indicated the involvement of chitinases. The expressed chitinases ChiA, ChiB and ChiC1 from S. marcescens GEI by recombinant Escherichia coli showed pathogenicity against the mites in the laboratory. These chitinases were active in a broad pH range (5-9) and the optimum temperatures were between 60 and 75 °C. Synergistic effects of ChiA and ChiB on the miticidal activity against V. destructor were observed. The workers of both honey bee species were not sensitive to the spraying and feeding chitinases. These results provided alternative control strategies for Varroa mites, by formulating chitinase agents and by constructing transgenetic honey bees.     

Effects of Bacillus thuringiensis toxin Cry1Ac and Beauveria bassiana on Asiatic corn borer (Lepidoptera: Crambidae)

In this study, interactions between Cry1Ac, a toxic crystal protein produced by Bacillus thuringiensis (Berliner), and Beauveria bassiana on the mortality and survival of Ostrinia furnacalis was evaluated in the laboratory. The results showed that Cry1Ac is toxic to O. furnacalis. Not only were larval growth and development delayed, but pupation, pupal weight and adult emergency also decreased when larvae were fed on artificial diet containing purified Cry1Ac toxin. When third instars O. furnacalis were exposed to combination of B. bassiana (1.8 × 10⁵, 1.8 × 10⁶ or 1.8 × 10⁷ conidia ml⁻¹) and Cry1Ac, (0.2 or 0.8 μg g⁻¹), the effect on mortality was additive, however, the combinations of sublethal concentrations showed antagonism between Cry1Ac (3.2 or 13 μg g⁻¹) and B. bassiana (1.8 × 10⁵ or 1.8 × 10⁶ conidia ml⁻¹). When neonates were reared on sublethal concentrations of Cry1AC until the third instar, and survivors exposed B. bassiana conidial suspension, such treatments showed additive effect on mortality of O. furnacalis except for the combination of Cry1Ac (0.2 μg g⁻¹) and B. bassiana (1.8 × 10⁶ conidia ml⁻¹) that showed antagonism.     

Rubrimonas shengliensis sp. nov. and Polymorphum gilvum gen. nov., sp. nov., novel members of Alphaproteobacteria from crude oil contaminated saline soil

Four bacterial strains were isolated from a crude oil contaminated saline soil in Shengli Oilfield, China. Strains SL014B-28A2^T and SL014B-80A1 were most closely related to Rubrimonas cliftonensis OCh 317^T, while strains SL003B-26A1^T and SL003B-26A2 were most closely related to but readily different from the species in the Pannonibacter-Labrenzia-Roseibium-Stappia cluster. The major fatty acids were C_18:1ω7c, C_16:0, C_18:0 and 11-Methyl C_18:1ω7c, and C_18:1ω7c, 11-Methyl C_18:1ω7c and C_18:0, respectively, for these two groups of isolates. Q-10 was the predominant ubiquinone. The G + C contents of genomic DNA of the four isolates were 67.9, 69.7, 65.6 and 65.6 mol%. Based on the polyphasic taxonomic characteristics, strains SL014B-28A2^T and SL014B-80A1 represented a novel species of the genus Rubrimonas, for which the name Rubrimonas shengliensis sp. nov. is proposed, with strain SL014B-28A2^T (=LMG 26072^T = CGMCC 1.9170^T) as the type strain. Isolates SL003B-26A1^T and SL003B-26A2 represented a novel genus and species of the family Rhodobacteraceae, for which the name Polymorphum gilvum gen. nov., sp. nov. is proposed, with strain SL003B-26A1^T (=LMG 25793^T = CGMCC 1.9160^T) as the type strain.     

Host age and pathogen dosage impact cyst morphogenesis in the invertebrate pathogenic alga Helicosporidium sp. (Chlorophyta: Trebouxiophyceae)

Helicosporidium sp. is a pathogenic alga that replicates in the hemolymph of various invertebrate hosts. Morphogenesis of the infectious life stage, the cyst, occurs in the infected host, but to date cannot be induced in vitro. Using larvae of the heterologous host Helicoverpa zea, we examined potential factors influencing pathogenicity and in vivo cyst production of the alga and the impact of infection on host survival. Factors tested were cyst dosage administered per os (ranging from 10² to 10⁵ cysts per larva) and host age at exposure (third, fourth, and fifth larval instar). Cyst production occurred between 7 and 13 days after treatment, regardless of host age at treatment. Increasing dosage increased both percent infection and mortality, but cyst production did not track the total infection response. Increasing host age at exposure mitigated dosage effects on infection and mortality and also elevated cyst production in later-treated larvae. Only the highest dosage produced a significant decrease in the overall time to death. Moderate cyst dosages and later host ages were most effective at regenerating Helicosporidium cysts.     

Definition of a novel symbiovar (sv. retamae) within Bradyrhizobium retamae sp. nov., nodulating Retama sphaerocarpa and Retama monosperma

In this paper we analyze through a polyphasic approach several Bradyrhizobium strains isolated in Spain and Morocco from root nodules of Retama sphaerocarpa and Retama monosperma. All the strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium lablabi CCBAU 23086^T, with 99.41% identity with respect to the strain Ro19^T. Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII were divergent in Ro19^T and B. lablabi CCBAU 23086^T, with identity values of 95.71%, 93.75% and 93.11%, respectively. These differences were congruent with DNA–DNA hybridization analysis that revealed an average of 35% relatedness between the novel species and B. lablabi CCBAU 23086^T. Also, differential phenotypic characteristics of the new species were found with respect to the already described species of Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose to classify the group of strains isolated from R. sphaerocarpa and R. monosperma as a novel species named Bradyrhizobium retamae sp. nov. (type strain Ro19^T = LMG 27393^T = CECT 8261^T). The analysis of symbiotic genes revealed that some of these strains constitute a new symbiovar within genus Bradyrhizobium for which we propose the name “retamae”, that mainly contains nodulating strains isolated from Retama species in different continents.