Synthesis and juvenile hormone activities of some new ether derivatives of hydroquinone

A new class of compounds, derived from hydroquinones, has been synthesised and bioassayed for juvenile hormone activity on yellow mealworm (Tenebrio molitor), confused flour beetle (Tribolium confusum), and mediterranean flour moth (Anagasta kuehniella). Structure-biological activity relationships of the most significant products are tabulated and discussed. Two of the new compounds, 1, 4-bis(5-chloropent-4-ynyloxy) benzene (I) and 1-(5-chloropent-4-ynyloxy)-4-phenoxybenzene (II), showed outstanding activity in morphogenetic assay with T. molitor, and good field persistence. Product II is of particular interest for the control of various Homopteran species, mosquitoes, stored product pests and fire ants.     

New chlorinated juvenile hormone analogues and their biological activity

In the search for new juvenile hormone analogues (JHAs), a general approach was chosen based on the substitution of the isoprene unit, that carries the epoxy function in the natural hormone JHIII, by 3,3,3-trichloropropyl, 2,2-dichlorocyclopropyl- methyl, 3,3-dichloroallyl, or 3-chloroprop-2-ynyl groups. Accordingly, several terpenoid aromatic JHAs were prepared with, or without, oxygen atoms in the aliphatic chain. Structure-biological activity relationships of the most active compounds toward the yellow mealworm (Tenebrio molitor) and the large cabbage white butterfly (Pieris brassicae) are given. With a view to preparing synthetically accessible analogues, some alkyl phenyl ethers were also made, which were not branched in the aliphatic chain and were related terminally to the most active compounds. They were only moderately active against insects when tested for morphogenetic activity, but exhibited a good field persistence. The occurrence of a moderate acaricidal activity among these analogues prompted the examination of several 1,4-disubstituted-phenyl derivatives. The ovicidal effectiveness of these products against a susceptible strain of the two-spotted spider mite, Tetranychus urticae, was ten or 100 times higher than that of the well known and highly potent JHAs.     

Molecular characterization of Fusarium oxysporum f.sp. cichorii pathogenic on chicory (Cichorium intybus)

Fusarium oxysporum is a ubiquitous soilborne ascomycete responsible for vascular wilt in many plant species worldwide. This species comprises more than 120 putative host-specific formae speciales capable of causing marked economic losses. In summer 2009, wilt symptoms, including chlorosis and poor development of the root system, were observed on cultivars of chicory (Cichorium intybus) in northern Italy. The causal agent isolated from symptomatic tissues in this case was identified as F. oxysporum on the basis of both morphological features and molecular analyses. In this work, we attempted to characterize the isolates of F. oxysporum from C. intybus by both biological and molecular approaches. Pathogenicity trials performed on five species of the Asteraceae family with isolates of F. oxysporum from C. intybus indicated that the pathogen has a unique host range, infecting chicory only. Neither lettuce nor endive, lawn daisy or Paris daisy developed the disease. Five cultivars within C. intybus species were tested, and the cv. ??Clio?? was the most susceptible. Phylogenetic analyses relative to the ribosomal intergenic spacer (IGS) and translation elongation factor 1-alpha (EF1-??) assigned isolates pathogenic to chicory to a single cluster, distinct from other pathogenic F. oxysporum. In light of these findings, we propose to designate this organism as Fusarium oxysporum f.sp. cichorii.     

Conventional and real-time PCR for the identification of Fusarium fujikuroi and Fusarium proliferatum from diseased rice tissues and seeds

Fusarium fujikuroi is a species of the Gibberella fujikuroi species complex (GFSC) and the causal agent of bakanae disease on rice. Even if F. fujikuroi is the most abundant Fusarium species found on rice, other species can also be isolated from rice, such as F. proliferatum. Multiple alignment of translation elongation factor (TEF) gene sequences of different Fusarium spp., showed a deletion of six nucleotides in F. fujikuroi sequence and a two nucleotide polymorphism in the same region of F. proliferatum sequence. These elements of variability were used to develop a conventional and Real-Time PCR assay for diagnosis. The species specific primer pairs (Fuji1F/TEF1R and Proli1F/TEF1R) gave a product of 179 and 188?bp for F. fujikuroi and F. proliferatum respectively. Primer specificity was confirmed by analyzing the DNA of the most representative species of the GFSC and 298 strains of Fusarium spp. isolated from rice plants and seeds in Italy. The specific primers were also successfully used to detect fungal presence directly from infected rice tissues and seeds, providing a rapid tool for the early detection of pathogen contamination.     

Effect of oil-based formulations of acaripathogenic fungi to control Rhipicephalus microplus ticks under laboratory conditions

The formulations of acaripathogenic fungi to control ticks have been widely studied. The present study evaluated the efficacy of oil-based formulations of Metarhizium anisopliae sensu lato (s.l.), isolate Ma 959, and Beauveria bassiana, isolate Bb 986, on different Rhipicephalus microplus stages, comparing the efficacy between aqueous suspensions and 10, 15 and 20% mineral oil formulations. Twelve groups were formed: one aqueous control group; three mineral oil control groups, at 10, 15 or 20%; two aqueous fungal suspensions of M. anisopliae s.l. or B. bassiana; and three formulations of M. anisopliae (s.l.) or B. bassiana containing 10, 15, and 20% mineral oil. To prepare aqueous suspensions and oily formulations, fungal isolates were cultivated on rice grains in polypropylene bags. The conidial suspensions and formulations had a concentration of 10(8)conidia/mL. Bioassays were repeated twice. After treatment, the following biological parameters of engorged females were evaluated: hatching percentage, egg production index, nutritional index, and percentage of tick control. The following parameters were evaluated in the bioassays with eggs: period of incubation, period of hatch, and hatching percentage. Mortality was evaluated in bioassays with larvae. M. anisopliae s.l. and B. bassiana oil-based formulations were more effective than aqueous suspensions against R. microplus eggs, larvae and engorged females, however, there was no significant difference between the three oil concentrations used. M. anisopliae s.l. and B. bassiana formulated in mineral oil reached 93.69% and 21.67% efficacy, respectively, while M. anisopliae s.l. and B. bassiana aqueous suspensions attained 18.70% and 1.72% efficacy, respectively. M. anisopliae s.l. oil-based formulations caused significant effects in all biological parameters of engorged females while B. bassiana oil-based formulations modified significantly the nutritional index only. Eggs treated with M. anisopliae s.l. and B. bassiana oil-based formulations showed hatching rates that decreased 102.5 and 3.65 times, respectively. In the bioassay with larvae, M. anisopliae s.l. oil-based formulations caused nearly 100% mortality five days after treatment, while larva treated with B. bassiana oil-based formulations reached 100% mortality at day 20 after treatment. Larva from oil-based control groups showed mortality at day 15 after treatment, which indicated a possible toxic effect of the oil for this R. microplus stage. The results showed that the fungal mineral oil formulations tested were more effective than the aqueous suspension. Oil-based formulations at 10, 15 and 20% enhanced the activity of M. anisopliae s.l. Ma 959, and B. bassiana Bb 986, isolates against R. microplus eggs, larvae, and engorged females tick. Mineral oil was effective as an adjuvant in formulations of M. anisopliae s.l., Ma 959, and B. bassiana, Bb 986, for the control of R. microplus under laboratory conditions.     

Phylogenetic and structural studies of a novel equine papillomavirus identified from aural plaques

Papillomaviruses (PVs) infect a wide range of animal species and show great genetic diversity. To date, excluding equine sarcoids, only three species of PVs were identified associated with lesions in horses: Equus caballus papillomavirus 1 (EcPV1-cutaneous), EcPV2 (genital) and EcPV3 (aural plaques). In this study, we identified a novel equine PV from aural plaques, which we designated EcPV4. Cutaneous samples from horses with lesions that were microscopically diagnosed as aural plaques were subjected to DNA extraction, amplification and sequencing. Rolling circle amplification and inverse PCR with specific primers confirmed the presence of an approximately 8 kb circular genome. The full-length EcPV4 L1 major capsid protein sequence has 1488 nucleotides (495 amino acids). EcPV4 had a sequence identity of only 53.3%, 60.2% and 51.7% when compared with the published sequences for EcPV1, EcPV2 and EcPV3, respectively. A Bayesian phylogenetic analysis indicated that EcPV4 clusters with EcPV2, but not with EcPV1 and EcPV3. Using the current PV classification system that is based on the nucleotide sequence of L1, we could not define the genus of the newly identified virus. Therefore, a structural analysis of the L1 protein was carried out to aid in this classification because EcPV4 cause lesion similar to the lesion caused by EcPV3. A comparison of the superficial loops demonstrated a distinct amino acid conservation pattern between EcPV4/EcPV2 and EcPV4/EcPV3. These results demonstrate the presence of a new equine PV species and that structural studies could be useful in the classification of PVs.     

Design and Synthesis of Pro-Apoptotic Compounds Inspired by Diatom Oxylipins

Oxylipins are a large and diverse family of fatty acid derivatives exhibiting different levels of oxidation of the carbon chain. They are involved in many biological functions in mammals, plants and diatoms. In this last group of organisms, they are suggested to play a role in the reproductive failure of copepod predators, showing clear pro-apoptotic effects on newborn nauplii. In this work, these compounds were tested for the ability to induce mitotic arrest in sea urchin embryos. We show for the first time that oxylipins have an increased efficacy in their corresponding methylated form. Natural oxylipins were also used as an inspiration for the rational design and synthesis of stable chemical analogs with apoptotic activity against tumor cell lines. This approach led to the synthesis of the linear C15-ketol (22) that was shown to induce apoptosis in human leukemia U-937 cells. These results are proof of the concept of the use of eco-physiological considerations as a platform to guide the search for novel drug candidates.     

Composition and Quantitation of Microalgal Lipids by ERETIC 1H NMR Method

Accurate characterization of biomass constituents is a crucial aspect of research in the biotechnological application of natural products. Here we report an efficient, fast and reproducible method for the identification and quantitation of fatty acids and complex lipids (triacylglycerols, glycolipids, phospholipids) in microalgae under investigation for the development of functional health products (probiotics, food ingredients, drugs, etc.) or third generation biofuels. The procedure consists of extraction of the biological matrix by modified Folch method and direct analysis of the resulting material by proton nuclear magnetic resonance (¹H NMR). The protocol uses a reference electronic signal as external standard (ERETIC method) and allows assessment of total lipid content, saturation degree and class distribution in both high throughput screening of algal collection and metabolic analysis during genetic or culturing studies. As proof of concept, the methodology was applied to the analysis of three microalgal species (Thalassiosira weissflogii, Cyclotella cryptica and Nannochloropsis salina) which drastically differ for the qualitative and quantitative composition of their fatty acid-based lipids.