Significance Inoculation of cereals with diazotrophic (N 2 -fixing) bacteria offers a sustainable alternative to the application of nitrogen fertilizers in agriculture. While natural diazotrophs have evolved multilayered regulatory mechanisms that couple N 2 fixation with assimilation of the product NH 3 and prevent release to plants, genetic modifications can permit excess production and excretion of NH 3 . However, a lack of stringent host-specificity for root colonization by the bacteria would allow growth promotion of target and nontarget plants species alike. Here, we exploit synthetic transkingdom signaling to establish plant host-specific control of the N 2 -fixation catalyst nitrogenase in Azorhizobium caulinodans occupying barley roots. This work demonstrates how partner-specific interactions can be established to avoid potential growth promotion of nontarget plants.

The essential oils of Thymus caespititius, T. camphoratus, T. capitellatus, T. carnosus, T. pulegioides, T. zygis subsp. zygis and T. zygis subsp. sylvestris collected in diff erent regions of Portugal, including the Azores islands, were evaluated for preventing lipid peroxidation and scavenging free radicals, and hydroxyl and superoxide anions. T. zygis subsp. zygis oil possessed the best capacity for preventing lipid peroxidation (IC 50 = 0.030 ± 0.003 mg/ml), immediately followed by the oils isolated from T. zygis subsp. sylvestris collected in diff erent regions of Portugal. IC 50 values ranged from 0.066 ± 0.003 to 0.087 ± 0.001 mg/ml in oils isolated from samples from Alcanena and Duas Igrejas, respectively. The oils isolated from T. zygis subsp. sylvestris, independent of the harvesting region, were shown to be the most eff ective for scavenging free radicals (0.358-0.453 mg/ml). The best capacity for scavenging hydroxyl radicals was found in the oils of T. camphoratus collected in Espartal and of T. caespititius of Lordelo, with IC 50 < 0.5 mg/ml. The low oil amount did not allow IC 50 values for the superoxide anion scavenging determination, therefore considering a unique oil concentration (5 mg/ml), the highest inhibition percentages (>50%) were registered with the essential oils of T. zygis subsp. sylvestris from Duas Igrejas and Covão do Coelho, and of T. capitellatus from Sines.

Pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, a serious threat to global forest populations of conifers, especially Pinus spp. A time-course study of the essential oils (EOs) of 2-year-old Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris following inoculation with the PWN was performed. The constitutive and nematode inoculation induced EOs components were analyzed at both the wounding or inoculation areas and at the whole plant level. The enantiomeric ratio of optically active main EOs components was also evaluated. External symptoms of infection were observed only in P. pinaster and P. sylvestris 21 and 15 days after inoculation, respectively. The EO composition analysis of uninoculated and unwounded plants revealed the occurrence of chemotypes for P. pinaster, P. halepensis and P. sylvestris, whereas P. pinea showed a homogenous EO composition. When whole plants were evaluated for EO and monoterpene hydrocarbon enantiomeric chemical composition, no relevant qualitative and quantitative differences were found. Instead, EO analysis of inoculated and uninoculated wounded areas revealed an increase of sesquiterpenes and diterpenic compounds, especially in P. pinea and P. halepensis, comparatively to healthy whole plants EOs.

Acetone was investigated and found to be an appropriate alternative to Triton X-100 as a solvent of essential oils in bioassays aimed to investigate their effects on pinewood nematode (Bursaphelenchus xylophilus) mortality. Therefore it was used as dilution agent to screen the effectiveness of fifty two essential oils against this pest. Thirteen essential oils were highly effective, resulting in more than 90% pinewood nematode mortality at 2 mg/mL, with six of them resulting in 100% mortality. LC100 values ranged between 0.50 mg/mL and 0.83 mg/mL for the essential oils of Origanum vulgare and Satureja montana, respectively. Essential oils were submitted to gas chromatography and gas chromatography-mass spectrometry analysis and their chemical composition established. Data from essential oils with 100% mortality at 2 mg/mL and other essential oils previously found to have LC100 ≤ 2 mg/mL was combined, their chemical profiles investigated by correspondences analysis plus automatic classification.

Assessment of plant-associative bacterial nitrogen (N) fixation is crucial for selection and development of elite diazotrophic inoculants that could be used to supply cereal crops with nitrogen in a sustainable manner. Although diazotrophic bacteria possess diverse oxygen tolerance mechanisms, most require a sub 21% oxygen environment to achieve optimal stability and function of the N-fixing catalyst nitrogenase. Consequently, assessment of N fixation is routinely carried out on “free-living” bacteria grown in the absence of a host plant and such experiments may not accurately divulge activity in the rhizosphere where the availability and forms of nutrients such as carbon and N, which are key regulators of N fixation, may vary widely. Here, we present a modified in situ acetylene reduction assay (ARA), utilizing the model cereal barley as a host to comparatively assess nitrogenase activity in diazotrophic bacteria. The assay is rapid, highly reproducible, applicable to a broad range of diazotrophs, and can be performed with simple equipment commonly found in most laboratories that investigate plant-microbe interactions. Thus, the assay could serve as a first point of order for high-throughput identification of elite plant-associative diazotrophs.

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The essential oils isolated from the vegetative aerial parts (mature branches with leaves) of 16 Eucalyptus species, grown in Mata Experimental do Escaroupim (Salvaterra de Magos, Portugal) were studied. The essential oils from E.