Factors Structuring the Epiphytic Archaeal and Fungal Communities in a Semi-arid Mediterranean Ecosystem.

The phyllosphere microbiome exerts a strong effect on plants' productivity, and its composition is determined by various factors. To date, most phyllosphere studies have focused on bacteria, while fungi and especially archaea have been overlooked. We studied the effects of plant host and season on the abundance and diversity of the epiphytic archaeal and fungal communities in a typical semi-arid Mediterranean ecosystem. We collected leaves in two largely contrasting seasons (summer and winter) from eight perennial species of varying attributes which could be grouped into the following: (i) high-canopy, evergreen sclerophyllοus shrubs with leathery leaves, and low-canopy, either semi-deciduous shrubs or non-woody perennials with non-leathery leaves, and (ii) aromatic and non-aromatic plants. We determined the abundance of epiphytic Crenarchaea, total fungi, Alternaria and Cladosporium (main airborne fungi) via q-PCR and the structure of the epiphytic archaeal and fungal communities via amplicon sequencing. We observed a strong seasonal effect with all microbial groups examined showing higher abundance in summer. Plant host and season were equally important determinants of the composition of the fungal community consisted mostly of Ascomycota, with Hypocreales dominating in winter and Capnodiales and Pleosporales in summer. In contrast, the archaeal community showed plant host driven patterns dominated by the Soil Crenarchaeotic Group (SCG) and Aenigmarchaeota. Plant habit and aromatic nature exhibited filtering effects only on the epiphytic fungal communities. Our study provides a first in-depth analysis of the key determinants shaping the phyllosphere archaeal and fungal communities of a semi-arid Mediterranean ecosystem.

Bacteria Affect Plant-Mite Interactions Via Altered Scent Emissions.

Epiphytic bacteria have been shown to affect the composition of volatiles released by plants and as a consequence the behavior of other organisms towards the plant, such as herbivores and/or pathogens. In this study, we explored the effects of inoculation with three bacterial strains, namely Pseudomonas syringae, Pantoea ananatis, and Pseudomonas putida, on the composition of leaf volatile organic compounds (VOCs) emitted by bean plants (Phaseolus vulgaris L.). In addition, we examined responses of the two-spotted spider mite (Tetranychus urticae) to VOCs by measuring leaf damage and oviposition of female adults after bacterial inoculation. Colonized bean plants emitted different VOCs depending on the bacterial inoculum. The quantities of volatiles 1-undecanol and (Z)-3-hexen-1-ol significantly increased after P. syringae inoculation, while methyl salicylate and anisole increased in response to P. ananatis. T. urticae females preferred control plants over plants inoculated with P. syringae or P. putida in olfactometer assays, while no particular preference was recorded in the presence of P. ananatis. Furthermore, leaf damage caused by spider mites was 3-fold lower in plants inoculated with P. syringae than in control plants and plants inoculated with P. ananatis. Subsequently, the number of eggs laid on leaves inoculated with P. syringae was significantly lower than on those inoculated with P. ananatis or on the control ones. Moreover, a significantly higher number of spider mites selected methyl salicylate odor source over 1-undecanol, in a two-choice bioassay. The results demonstrate the bacterial involvement in plant-arthropod interactions and suggest further investigation on the potential use of bacteria as biocontrol agents in agriculture.

Are leaf glandular trichomes of oregano hospitable habitats for bacterial growth?

Phyllospheric bacteria were isolated from microsites around essential-oil-containing glands of two oregano (Origanum vulgare subsp. hirtum) lines. These bacteria, 20 isolates in total, were subjected to bioassays to examine their growth potential in the presence of essential oils at different concentrations. Although there were qualitative and quantitative differences in the essential oil composition between the two oregano lines, no differences were recorded in their antibacterial activity. In disk diffusion bioassays, four of the isolated strains could grow almost unrestrained in the presence of oregano oil, another five proved very sensitive, and the remaining 11 showed intermediate sensitivity. The strain least inhibited by oregano essential oil was further identified by complete16s rRNA gene sequencing as Pseudomonas putida. It was capable of forming biofilms even in the presence of oregano oil at high concentrations. Resistance of P. putida to oregano oil was further elaborated by microwell dilution bioassays, and its topology on oregano leaves was studied by electron microscopy. When inoculated on intact oregano plants, P. putida was able not only to colonize sites adjacent to essential oil-containing glands, but even to grow intracellularly. This is the first time that such prolific bacterial growth inside the glands has been visually observed. Results of this study further revealed that several bacteria can be established on oregano leaves, suggesting that these bacteria have attributes that allow them to tolerate or benefit from oregano secondary metabolites.

Bacterial colonization of the phyllosphere of mediterranean perennial species as influenced by leaf structural and chemical features.

In this study, we assessed various leaf structural and chemical features as possible predictors of the size of the phyllosphere bacterial population in the Mediterranean environment. We examined eight perennial species, naturally occurring and coexisting in the same area, in Halkidiki (northern Greece). They are Arbutus unedo, Quercus coccifera, Pistacia lentiscus, and Myrtus communis (evergreen sclerophyllous species), Lavandula stoechas and Cistus incanus (drought semi-deciduous species), and Calamintha nepeta and Melissa officinalis (non-woody perennial species). M. communis, L. stoechas, C. nepeta, and M. officinalis produce essential oil in substantial quantities. We sampled summer leaves from these species and (1) estimated the size of the bacterial population of their phyllosphere, (2) estimated the concentration of different leaf constituents, and (3) studied leaf morphological and anatomical features and expressed them in a quantitative way. The aromatic plants are on average more highly colonized than the other species, whereas the non-woody perennials are more highly colonized than the woody species. The population size of epiphytic bacteria is positively correlated with glandular and non-glandular trichome densities, and with water and phosphorus contents; it is negatively correlated with total phenolics content and the thickness of the leaf, of the mesophyll, and of the abaxial epidermis. No correlation was found with the density of stomata, the nitrogen, and the soluble sugar contents. By regression tree analysis, we found that the leaf-microbe system can be effectively described by three leaf attributes with leaf water content being the primary explanatory attribute. Leaves with water content >73% are the most highly colonized. For leaves with water content <73%, the phosphorus content, with a critical value of 1.34 mg g(-1) d.w., is the next explanatory leaf attribute, followed by the thickness of the adaxial epidermis. Leaves higher in phosphorus (>1.34 mg g(-1) d.w.) are more colonized, and leaves with the adaxial epidermis thicker than 20.77 microm are the least colonized. Although these critical attributes and values hold true only within the Mediterranean ecosystem studied and the range of observations taken, they are important because they provide a hypothesis to be tested in other Mediterranean ecosystems and other biomes. Such comparative studies may give insight as to the general properties governing the leaf-microbe system.

Hypervalent iodine compounds as potent antibacterial agents against ice nucleation active (INA) Pseudomonas syringae.

Twenty-three hypervalent iodine compounds belonging to aryliodonium salts, 1, aryliodonium ylides, 2, and (diacyloxyiodo)arenes, 3, were tested for their antibacterial activities against ice nucleation active (INA) Pseudomonas syringae, and the MIC and EC(50) values were determined. All of the compounds examined caused a dose-dependent decrease in bacterial growth rates. Aryliodonium salts, especially those with electron-withdrawing groups, exhibit higher antibacterial activities with MIC = 8-16 ppm, whereas the nature of the anion does not seem to affect the activities of the diaryliodonium salts.