Katerina Karamanoli

Bacterial colonization of the phyllosphere of nineteen plant species and antimicrobial activity of their leaf secondary metabolites against leaf associated bacteria

Summary.The scope of this work was to examine whether leaf constitutive secondary metabolites play a role in determining bacterial colonization of the phyllosphere. To this aim, we surveyed nineteen native or cultivated plant species that share a common bacterial pool in a North Mediterranean area, and estimated the size of total and ice nucleation active (INA) bacterial populations on their leaves. Large differences in the colonization of their phyllosphere were found; the population size of epiphytic bacteria ranged from 7.5 × 102 to 1 × 106 CFU/g fresh weight, in eucalypt and celery, respectively. Species native in Mediterranean-type climate areas, particularly those belonging to the group of aromatic plants, are characterized by scarce presence of INA bacteria. The antibacterial activity of essential oils, surface phenolics and leaf tissue extracts was also estimated against the INA strains P. syringae and E. herbicola, isolated from two of these plant species. E. herbicola proved more sensitive than P. syringae. Of the species examined, oregano [Origanum vulgare L. subsp. hirtum (Link.) Ietswaart], an aromatic plant, had the highest antimicrobial activity, whereas six species showed no activity at all. Further experiments were performed with oregano and bean (Phaseolus vulgaris L.) that represent two extremes in their secondary metabolite content. Both plants were inoculated with P. syringae. By the end of incubation, the bacterial population on bean plants was about 100 times higher than that on oregano leaves. Scanning electron micrographs showed that bacterial growth on oregano leaves was confined to sites away from glandular hairs. Results from the bacterial colonization survey together with those from the toxicity tests showed that all species rich in antibacterial secondary metabolites harbored low leaf bacterial populations. These results provide substantial evidence that leaf secondary metabolites function as constitutive defense chemicals against microbial invasions. However, the fact that species with non- or moderately active leaf secondary metabolites are not always highly colonized suggests mediation of other unknown factors, the contribution of which requires further investigation.

Exploring Biodiversity in the Bacterial Community of the Mediterranean Phyllosphere and its Relationship with Airborne Bacteria

We studied the structure and diversity of the phyllosphere bacterial community of a Mediterranean ecosystem, in summer, the most stressful season in this environment. To this aim, we selected nine dominant perennial species, namely Arbutus unedo, Cistus incanus, Lavandula stoechas, Myrtus communis, Phillyrea latifolia, Pistacia lentiscus, Quercus coccifera (woody), Calamintha nepeta, and Melissa officinalis (herbaceous). We also examined the extent to which airborne bacteria resemble the epiphytic ones. Genotype composition of the leaf and airborne bacteria was analysed by using denaturing gradient gel electrophoresis profiling of a 16S rDNA gene fragment; 75 bands were cloned and sequenced corresponding to 28 taxa. Of these, two were found both in the air and the phyllosphere, eight only in the air, and the remaining 18 only in the phyllosphere. Only four taxa were found on leaves of all nine plant species. Cluster analysis showed highest similarity for the five evergreen sclerophyllous species. Aromatic plants were not grouped all together: the representatives of Lamiaceae, bearing both glandular and non-glandular trichomes, formed a separate group, whereas the aromatic and evergreen sclerophyllous M. communis was grouped with the other species of the same habit. The epiphytic communities that were the richest in bacterial taxa were those of C. nepeta and M. officinalis (Lamiaceae). Our results highlight the remarkable presence of lactic acid bacteria in the phyllosphere under the harsh conditions of the Mediterranean summer, the profound dissimilarity in the structure of bacterial communities in phyllosphere and air, and the remarkable differences of leaf microbial communities on neighbouring plants subjected to similar microbial inocula; they also point to the importance of the leaf glandular trichome in determining colonization patterns.

Inhibitory and stimulatory effects of essential oils and individual monoterpenoids on growth and sporulation of four soil-borne fungal isolates of Aspergillus terreus, Fusarium oxysporum, Penicillium expansum, and Verticillium dahliae

The effect of essential oils and individual monoterpenoids on soil-borne fungi, in pure and mixed cultures, in growth media and in the soil environment, was investigated. Essential oils were extracted from lavender (Lavandula stoechas), oregano (Origanum vulgare subsp. hirtum), sage (Salvia fruticosa) and spearmint (Mentha spicata). The monoterpenoids tested were fenchone, carvacrol, 1,8-cineole, carvone, α-pinene and terpinen-4-ol. Their effect was examined on growth and sporulation of Aspergillus terreus, Fusarium oxysporum, Penicillium expansum and Verticillium dahliae isolated from an organic cultivation of tomato. All tested essential oils and individual monoterpenoids inhibited mycelial growth in all fungi and conidial production in most fungi. The strongest inhibitory activity on mycelial growth was exhibited by oregano and spearmint oils and by carvacrol and carvone, respectively their main constituents. The inhibitory activity was clearly fungistatic in A. terreus and F. oxysporum but fungicidal in V. dahliae. On sporulation, clearly stimulatory effects were observed alongside inhibitory ones. Conidial production was always promoted by α-pinene in P. expansum and by sage oil in F. oxysporum. At certain dosages it was promoted by cineole and carvone in F. oxysporum, and by lavender oil in A. terreus and V. dahliae. Experiments with carvone and carvacrol against mixed fungal cultures in a soil environment showed that V. dahliae was the most sensitive and A. terreus the most tolerant of the four fungi. Our results demonstrate strong but divergent effects and selectivity of action of the lower terpenoids on fungal strains that can become serious pests of tomato. Of special importance is the complete inhibition of growth and conidial production of V. dahliae, a pathogen otherwise very resistant to chemical control.

Abundance and diversity of the phyllosphere bacterial communities of Mediterranean perennial plants that differ in leaf chemistry

Summary.We studied the epiphytic bacterial communities of the summer leaves of eight perennial species naturally occurring in a Mediterranean ecosystem. The species differ in essential-oil content (from rich in essential oil to non-producers) and composition, and also in life form (from herbaceous species to tall shrubs). We compared the epiphytic bacterial communities on the basis of (i) their abundance, (ii) their metabolic profile (derived by use of the BIOLOG Ecoplate system) and (iii) richness and diversity of substrates that they use, as a measure of functional diversity. Among all species, the aromatic Melissa officinalis was the most abundantly colonized. The bacterial communities on the leaves of the aromatic Myrtus communis, Calamintha nepeta and Melissa officinalis, and also of Cistus incanus catabolized all 31 substrates offered; those on the evergreen-sclerophyllous species, Arbutus unedo and Quercus coccifera, catabolized only 14 and 17 substrates, respectively. Carbohydrates were consistently used abundantly by all communities, whereas carboxylic acids were most variably used. On average, the group of aromatic plants scored higher regarding bacterial abundance, and richness and diversity of substrates used by the bacterial communities on their leaves; the lowest values for both substrate-use indices were recorded in A. unedo. Bacterial abundance or richness or diversity of substrates used did not vary with leaf oil content. Abundance was positively correlated with both substrate-use indices. Results support claims that the antimicrobial effects of essential oils are not exerted so much under natural conditions as reports based on biassays with pathogens usually show. Although essential oils play a part in the microbial colonization of the phyllosphere, it is not likely that inhibition of phyllosphere bacteria is essential oils’ primary role, at least in the Mediterranean environment.

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.

Deregulation of apoplastic polyamine oxidase affects development and salt response of tobacco plants

Polyamine (PA) homeostasis is associated with plant development, growth and responses to biotic/abiotic stresses. Apoplastic PA oxidase (PAO) catalyzes the oxidation of PAs contributing to cellular homeostasis of reactive oxygen species (ROS) and PAs. In tobacco, PAs decrease with plant age, while apoplastic PAO activity increases. Our previous results with young transgenic tobacco plants with enhanced/reduced apoplastic PAO activity (S-ZmPAO/AS-ZmPAO, respectively) established the importance of apoplastic PAO in controlling tolerance to short-term salt stress. However, it remains unclear if the apoplastic PAO pathway is important for salt tolerance at later stages of plant development. In this work, we examined whether apoplastic PAO controls also plant development and tolerance of adult plants during long-term salt stress. The AS-ZmPAO plants contained higher Ca2+ during salt stress, showing also reduced chlorophyll content index (CCI), leaf area and biomass but taller phenotype compared to the wild-type plants during salt. On the contrary, the S-ZmPAO had more leaves with slightly greater size compared to the AS-ZmPAO and higher antioxidant genes/enzyme activities. Accumulation of proline in the roots was evident at prolonged stress and correlated negatively with PAO deregulation as did the transcripts of genes mediating ethylene biosynthesis. In contrast to the strong effect of apoplastic PAO to salt tolerance in young plants described previously, the effect it exerts at later stages of development is rather moderate. However, the different phenotypes observed in plants deregulating PAO reinforce the view that apoplastic PAO exerts multifaceted roles on plant growth and stress responses. Our data suggest that deregulation of the apoplastic PAO can be further examined as a potential approach to breed plants with enhanced/reduced tolerance to abiotic stress with minimal associated trade-offs.

Ethylene –dependent and –independent superficial scald resistance mechanisms in ‘Granny Smith’ apple fruit

Superficial scald is a major physiological disorder of apple fruit (Malus domestica Borkh.) characterized by skin browning following cold storage; however, knowledge regarding the downstream processes that modulate scald phenomenon is unclear. To gain insight into the mechanisms underlying scald resistance, ‘Granny Smith’ apples after harvest were treated with diphenylamine (DPA) or 1-methylcyclopropene (1-MCP), then cold stored (0 °C for 3 months) and subsequently were ripened at room temperature (20 °C for 8 days). Phenotypic and physiological data indicated that both chemical treatments induced scald resistance while 1-MCP inhibited the ethylene-dependent ripening. A combination of multi-omic analysis in apple skin tissue enabled characterization of potential genes, proteins and metabolites that were regulated by DPA and 1-MCP at pro-symptomatic and scald-symptomatic period. Specifically, we characterized strata of scald resistance responses, among which we focus on selected pathways including dehydroabietic acid biosynthesis and UDP-D-glucose regulation. Through this approach, we revealed scald-associated transcriptional, proteomic and metabolic signatures and identified pathways modulated by the common or distinct functions of DPA and 1-MCP. Also, evidence is presented supporting that cytosine methylation-based epigenetic regulation is involved in scald resistance. Results allow a greater comprehension of the ethylene–dependent and –independent metabolic events controlling scald resistance.

Postharvest responses of sweet cherry fruit and stem tissues revealed by metabolomic profiling

Sweet cherry, a non-climacteric and highly perishable fruit, is usually cold-stored during post-harvest period to prevent senescence; therefore, metabolic profiling in response to cold storage in sweet cherry is of economic and scientific interest. In the present work, metabolic analysis was performed in fruit and stem tissues to determine the metabolic dynamics associated with cold storage in response to 1-methylcyclopropene (1-MCP), an ethylene-action inhibitor, and modified atmosphere packaging (MAP). Fruit (cv. Regina) following harvest were treated with 1-MCP and then cold-stored (0 °C, relative humidity 95%) for 1 month in the presence or in the absence of MAP and subsequently maintained at 20 °C for up to 2 days. Physiological analysis suggested that cold storage stimulated anthocyanin production, respiration rate and stem browning. Cherry stem exposed to 1-MCP displayed senescence symptoms as demonstrated by the higher stem browning and the lower stem traction force while MAP treatment considerably altered these features. The metabolic profile of fruits and stems just following cold storage was distinctly different from those analyzed at harvest. Marked tissue-specific differences were also detected among sweet cherries exposed to individual and to combined 1-MCP and MAP treatments, notably for amino acid biosynthesis. The significance of some of these metabolites as cold storage hallmarks is discussed in the context of the limited knowledge on the 1-MCP and MAP response mechanisms at the level of cherry fruit and stem tissues. Overall, this study provides the first steps toward understanding tissue-specific postharvest behavior in sweet cherry under various conditions.

Genotype, ultraviolet irradiation, and harvesting time interaction effects on secondary metabolites of whole lettuce and browning of fresh-cut product

ABSTRACT This study evaluated the importance of three factors (genotype, ultraviolet [UV] radiation and harvest time) and their interaction on phenolics, tannins, flavonoids and antioxidant activity of ‘Paris Island’ (romaine type) and ‘Sanguine’ (red leaf type) lettuce plants as well as on the severity of browning of their fresh-cut product. Ten genotypes had significant effect on secondary metabolite accumulation and higher levels were found in ‘Sanguine’, while no interaction effect was found among all the studied factors. Therefore, a new analysis of variance was performed for each genotype separately. The results showed that in ‘Paris Island’ the time of harvest had a significant effect on all measured parameters with higher levels for plants from the night harvest, while UV radiation had little effect on the plants’ biochemistry. On the contrary, in ‘Sanguine’, UV radiation significantly affected the concentration of secondary metabolites and antioxidant capacity, with higher levels for plants grown under UV-open film. Browning evaluation of fresh-cut product derived from the above plants showed that factors affecting secondary metabolite concentration in whole plants at harvest did not have a similar effect. The findings indicate that there are appropriate treatments, for each genotype, that could achieve high production of secondary metabolites and improve their antioxidant properties.

Underexpression of apoplastic polyamine oxidase improves thermotolerance in Nicotiana tabacum

Polyamines (PAs) and hydrogen peroxide (H2O2), the product of PA oxidation by polyamine oxidase (PAO), are potential players affecting plant growth, development and responses to abiotic/biotic stresses. Genetically modified Nicotiana tabacum plants with altered PA/H2O2 homeostasis due to over/underexpression of the ZmPAO gene (S-ZmPAO/AS-ZmPAO, respectively) were assessed under heat stress (HS). Underexpression of ZmPAO correlates with increased thermotolerance of the photosynthetic machinery and improved biomass accumulation, accompanied by enhanced levels of the enzymatic and non-enzymatic antioxidants, whereas ZmPAO overexpressors exhibit significant impairment of thermotolerance. These data provide important clues on PA catabolism/H2O2/thermotolerance, which merit further exploitation.