CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS OF LAVANDULA SPP.

Lavenders belong to the family Labiatae and represent some of the most popular medicinal plants of great economic importance. Their essential oils are important for the perfume, cosmetic, flavouring and pharmaceutical industries. However, despite its popularity, and the long tradition of use, biological properties of the various Lavandula species are not yet been well sustained by scientific or clinical studies and some available data being inconclusive and controversial [1]. Although Lavandula spp. have similar ethnobotanical properties, however, chemical composition and therapeutic uses differ from different species and main composition of essential oils showed differences with species and with the region were they grow [1,2,3]. L. stoechas L. subsps. luisieri (Rozeira) Rozeira. L. pedunculata (Mill.) Cav. and L. viridis L’Hér are endemic to the Iberian Peninsula, widespread in the South of Portugal, namely in Alentejo and Algarve. In our work, essential oils from the stems or leaves from wild grown plants of L. luisieri (Alentejo), L. pedunculata (Alentejo) and L. viridis (Algarve), were extracted by hydrodistillation and analyzed by GC-FID. Antimicrobial activity was evaluated by solid diffusion disk assay and minimal inhibitory concentration (MIC) against pathogenic Gram-positive and Gram-negative bacteria and food spoilage fungi.

Antioxidant,antimicrobial and toxicological properties of Schinus molle L. essential oils

Ethnopharmacological relevance: Schinus molle L. has been used in folk medicine as antibacterial, antiviral, topical antiseptic, antifungal, antioxidant, anti-inflammatory, anti-tumoural as well as antispasmodic and analgesic; however, there are few studies of pharmacological and toxicological properties of S. molle essential oils. Aim of the study: The aim of this study was to evaluate the antioxidant and antimicrobial activities of S. molle leaf and fruit essential oils, correlated with their chemical composition and evaluate their acute toxicity. Materials and methods: The chemical composition of S. molle leaf and fruit essential oils were evaluated by GC-FID and GC-MS. Antioxidant properties were determined using the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical and β-carotene/linoleic acid methods. Antimicrobial properties were evaluated by the agar disc diffusion method and minimal inhibitory concentration assay. Toxicity in Artemia salina and acute toxicity with behavioural screening in mice were evaluated. Results: The dominant compounds found in leaf and fruit essential oils (EOs) were monoterpene hydrocarbons, namely -phellandrene, β-phellandrene, β-myrcene, limonene and α-pinene. EOs showed low scavenging antioxidant activity by the DPPH free radical method and a higher activity by the β-carotene/linoleic acid method. Antimicrobial activity of EOs was observed for Gram+, Gram– pathogenic bacteria and food spoilage fungi. EOs showed cytotoxicity for Artemia salina and lower toxicity in Swiss mice. Conclusions: The result showed that EOs of leaves and fruits of S. molle demonstrated antioxidant and antimicrobial properties, suggesting their potential use in food or pharmaceutical industries.

ANTIOXIDANT, ANTIMICROBIAL AND TOXICOLOGICAL PROPERTIES OF SCHINUS MOLLE L. ESSENTIAL OILS

Schinus molle L. is commonly known as pink pepper or American pepper, of Anacardiaceae family, from subtropical regions of South America, introduced and naturalized in South Europe, including Portugal. In folk medicine, plant extracts and essential oil has related as having antibacterial, antiviral, antifungal, anti-inflammatory, antitumoral, antispasmodic, analgesic and antidepressive properties. The aim of present study was to evaluate the chemical composition and biological activities of essential oil extracted from leaves and fruits of S. molle. For this purpose, the essential oils were analyzed by gas chromatography (GC/FID) and antioxidant properties were evaluated by the free radical DPPH and by system β-carotene/linoleic acid methods. The antimicrobial activities were screened against pathogenic bacteria and fungi and food spoiling fungi by the disc diffusion assay and minimal inhibitory concentration (MIC) was determined for sensitive strains. Toxicity of essential oils were carried out by the brine shrimp mortality test (EC50) and acute lethal dose (DL50) determination after oral administration in Swiss mice The major components in leaf essential oil were α-phellandrene, β-phellandrene and limonene, while myrcene, α-phellandrene and 1,8-cineole are the main components in the fruit essential oil. The essential oils of leaf and fruit of S. molle showed antioxidant activity through the two mechanisms: the ability to capture free radicals and protection of lipid peroxidation. These oils exhibited also a broad microbial activity spectrum, against pathogenic bacteria Gram-positive and Gram-negative and Candida spp. The fruit essential oil showed high cytotoxicity against Artemia salina. Essential oils of leaves and fruits of S. molle showed significant antioxidant and microbial properties, so the studies continue to clarify more in deep its toxicity, including hepatotoxicity and nephrotoxicity, and to evaluate its medicinal or nutraceutical potential.

Detection of virulence-associated genes in pathogenic and commensal avian Escherichia coli isolates

Poultry colibacillosis due to Avian Pathogenic Escherichia coli (APEC) is responsible for several extra-intestinal pathological conditions, leading to serious economic damage in poultry production. The most commonly associated pathologies are airsacculitis, colisepticemia, and cellulitis in broiler chickens, and salpingitis and peritonitis in broiler breeders. In this work a total of 66 strains isolated from dead broiler breeders affected with colibacillosis and 61 strains from healthy broilers were studied. Strains from broiler breeders were typified with serogroups O2, O18, and O78, which are mainly associated with disease. The serogroup O78 was the most prevalent (58%). All the strains were checked for the presence of 11 virulence genes: 1) arginine succinyltransferase A (astA); ii) E. coli hemeutilization protein A (chuA); iii) colicin V A/B (cvaA/B); iv) fimbriae mannose-binding type 1 (fimC); v) ferric yersiniabactin uptake A (fyuA); vi) iron-repressible high-molecular-weight proteins 2 (irp2); vii) increased serum survival (iss); viii) iron-uptake systems of E. coli D (iucD); ix) pielonefritis associated to pili C (papC); x) temperature sensitive haemaglutinin (tsh), and xi) vacuolating autotransporter toxin (vat), by Multiplex-PCR. The results showed that all genes are present in both commensal and pathogenic E. coli strains. The iron uptake-related genes and the serum survival gene were more prevalent among APEC. The adhesin genes, except tsh, and the toxin genes, except astA, were also more prevalent among APEC isolates. Except for astA and tsh, APEC strains harbored the majority of the virulence-associated genes studied and fimC was the most prevalent gene, detected in 96.97 and 88.52% of APEC and AFEC strains, respectively. Possession of more than one iron transport system seems to play an important role on APEC survival.

Adventitious rooting of conifers: influence of biological factors

Vegetative propagation of superior conifer trees can be achieved e.g. through rooted cuttings or rooted microshoots, the latter predominantly through in vitro tissue culture. Both techniques are used to achieve rapid multiplication of trees with favorable genetic combinations and to capture a large proportion of the genetic diversity in a single generation cycle. However, adventitious rooting of shoots (cuttings) is often not efficient due to various problems such as scarcity of roots and cessation of their growth, both of which limit the application of vegetative propagation in some conifer species. Many factors are involved in the adventitious rooting of shoots including physical and chemical ones such as plant growth regulators, carbohydrates, light quality, temperature and rooting substrates or media (reviewed by Ragonezi et al. 2010). The focus of this review is on biological factors, such as inoculations with Agrobacterium rhizogenes, plant- growth-promoting rhizobacteria and other endophytes, and mycorrhizal fungi, which were found to stimulate adventitious rooting. These microorganisms could contribute not only to adventitious root development but also help in protecting conifer plants against pathogenic microorganisms, facilitate acclimation and transplanting, and contribute to more sustainable, chemical-free forests.

DETECTION OF SPORES AND HYPHAE OF ARTWORKS’ BIODETERIOGENIC FILAMENTOUS FUNGI BY RNA-FISH

Filamentous fungi are a threat to the conservation of Cultural Heritage. Thus, detection and identification of viable filamentous fungi are crucial for applying adequate Safeguard measures. RNA-FISH protocols have been previously applied with this aim in Cultural Heritage samples. However, only hyphae detection was reported in the literature, even if spores and conidia are not only a potential risk to Cultural Heritage but can also be harmful for the health of visitors, curators and restorers. Thus, the aim of this work was to evaluate various permeabilizing strategies for their application in the detection of spores/conidia and hyphae of artworks’ biodeteriogenic filamentous fungi by RNA-FISH. Besides of this, the influence of cell aging on the success of the technique and on the development of fungal autofluorescence (that could hamper the RNA-FISH signal detection) were also investigated. Five common biodeteriogenic filamentous fungi species isolated from biodegradated artworks were used as biological model: Aspergillus niger, Cladosporium sp, Fusarium sp, Penicillium sp. and Exophialia sp. Fungal autofluorescence was only detected in cells harvested from Fusarium sp, and Exophialia sp. old cultures, being aging-dependent. However, it was weak enough to allow autofluorescence/RNA-FISH signals distinction. Thus, autofluorescence was not a limitation for the application of RNA-FISH for detection of the taxa investigated. All the permeabilization strategies tested allowed to detect fungal cells from young cultures by RNA-FISH. However, only the combination of paraformaldehyde with Triton X-100 allowed the detection of conidia/spores and hyphae of old filamentous fungi. All the permeabilization strategies failed in the Aspergillus niger conidia/spores staining, which are known to be particularly difficult to permeabilize. But, even in spite of this, the application of this permeabilization method increased the analytical potential of RNA FISH in Cultural Heritage biodeterioration. Whereas much work is required to validate this RNA-FISH approach for its application in real samples from Cultural Heritage it could represent an important advance for the detection, not only of hyphae but also of spores and conidia of various filamentous fungi taxa by RNA-FISH.

Evidences for an opportunistic and endophytic lifestyle of the Bursaphelenchus xylophilus -associated bacteria Serratia marcescens PWN146 isolated from wilting Pinus pinaster

Pine wilt disease (PWD) results from the interaction of three elements: the pathogenic nematode, Bursaphelenchus xylophilus; the insect-vector, Monochamus sp.; and the host tree, mostly Pinus species. Bacteria isolated from B. xylophilus may be a fourth element in this complex disease. However, the precise role of bacteria in this interaction is unclear as both plant-beneficial and as plant-pathogenic bacteria may be associated with PWD. Using whole genome sequencing and phenotypic characterization, we were able to investigate in more detail the genetic repertoire of Serratia marcescens PWN146, a bacterium associated with B. xylophilus. We show clear evidence that S. marcescens PWN146 is able to withstand and colonize the plant environment, without having any deleterious effects towards a susceptible host (Pinus thunbergii), B. xylophilus nor to the nematode model C. elegans. This bacterium is able to tolerate growth in presence of xenobiotic/organic compounds, and use phenylacetic acid as carbon source. Furthermore, we present a detailed list of S. marcescens PWN146 potentials to interfere with plant metabolism via hormonal pathways and/or nutritional acquisition, and to be competitive against other bacteria and/or fungi in terms of resource acquisition or production of antimicrobial compounds. Further investigation is required to understand the role of bacteria in PWD. We have now reinforced the theory that B. xylophilus-associated bacteria may have a plant origin.