Helen-Isis A. Constantinidou

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.

Seasonal and diurnal photosynthetic responses of two gerbera cultivars to different substrates and heating systems

Abstract The simultaneous, long-term effects of three substrates (zeolite, perlite and their 1:1 mixture) and of two heating systems (lower and upper) upon the diurnal changes in photosynthesis of two gerbera cultivars (Cyprus and Heart Breaker) were investigated at different times of year. ‘Cyprus’ revealed higher net CO2 assimilation rate and stomatal conductance than ‘Heart Breaker’, while transpiration rate was almost the same. Based on both stomatal conductance and transpiration rate, the zeolite–perlite mixture substrate exerted a more beneficial effect on the soil–plant water system. No change in chlorophyll levels, either time-wise or between gerbera cultivars, was observed in response to different substrates and heating systems. Diurnal net CO2 assimilation rate, followed only for ‘Cyprus’, was reduced significantly from morning to late afternoon, possibly as a result of stomatal conductance reduction. The practical outcome with respect to the combined effect of different substrates, heating systems and cultivars was that ‘Cyprus’ grown on the mixture medium and subjected to an upper heating system (i.e. with heating pipes installed at the culture bag level) exhibited the best physiological responses.

Effect of different strength of medium on organogenesis, phenolic accumulation and antioxidant activity of spearmint (Mentha spicata L.).

We investigated the effect of inorganic salt concentration on the in vitro organogenesis of spearmint (Mentha spicata L.) by using Murashige and Skoog culture medium of different strength (full, half- and quarter strength). The highest number of shoots and roots induced per explant (3.5 and 10, respectively), as well as the maximum average shoot length (16 cm) was observed on half strength medium. The maximum leaf number (35 per explant) and average root length (7 cm) were associated with regeneration on full medium. The highest average phenolic content was observed on shoot extracts (7.20 mg/g f.w.) and root extracts (5.93 mg/g f.w.) cultured onto quarter and half strength medium, respectively. The strength of the culture medium was inversely correlated with the antioxidant activity of plant extracts. Therefore, for spearmint organogenesis, half strength MS medium offers a compromise between optimum growth in vitro and antioxidant phenolic accumulation.

Silencing S-Adenosyl-L-Methionine Decarboxylase (SAMDC) in Nicotiana tabacum Points at a Polyamine-Dependent Trade-Off between Growth and Tolerance Responses

Polyamines (PAs) are nitrogenous molecules that are indispensable for cell viability and with an agreed-on role in the modulation of stress responses. Tobacco plants with downregulated SAMDC (AS-SAMDC) exhibit reduced PAs synthesis but normal levels of PA catabolism. We used AS-SAMDC to increase our understanding on the role of PAs in stress responses. Surprisingly, at control conditions AS-SAMDC plants showed increased biomass and altered developmental characteristics, such as increased height and leaf number. On the contrary, during salt stress AS-SAMDC plants showed reduced vigor when compared to the WT. During salt stress, the AS-SAMDC plants although showing compensatory readjustments of the antioxidant machinery and of photosynthetic apparatus, they failed to sustain their vigor. AS-SAMDC sensitivity was accompanied by inability to effectively control H2O2 levels and concentrations of monovalent and divalent cations. In accordance with these findings, we suggest that PAs may regulate the trade-off between growth and tolerance responses.

Pronamide Effects on Physiology and Yield of Sugar Beet

Abstract Field experiments were conducted in northern Greece during 2001 and repeated in 2002 and 2004 to evaluate the effects of pronamide on sugar beet. Total leaf area, leaf area index (LAI), leaf and root dry weights, photosynthetic yield (quantum yield of photochemical energy conversion in photosystem II), chlorotic index, and yield components of sugar beet were monitored after pronamide application. Three sugar beet cultivars, ‘Avantage’, ‘Dorothea’, and ‘Bianca’, requiring short, intermediate, and long vegetative periods, respectively, were subjected to treatment. Pronamide was applied on sugar beet either as a double application of 0.63 kg ai ha−1 at the two- to four-leaf and 0.63 kg ai ha−1 at the four- to six-leaf stage or as a single application of 1.26 kg ai ha−1 performed at the latter leaf stage. Both application procedures were combined with a split application of phenmedipham at 0.04 kg ai ha−1 plus desmedipham at 0.04 kg ai ha−1 plus metamitron at 0.70 kg ai ha−1 plus ethofumesate at 0.10 kg ai ha−1 plus mineral oil at 0.50 L ha−1 applied POST at the cotyledon–to–two-leaf as well as at the four-leaf growth stages. Pronamide (both single and double application) initially caused chlorosis and reduction of sugar beet growth. LAI and photosynthetic yield were also significantly affected for a 2-mo period following the final application, after which the negative effects caused by pronamide were ameliorated. At harvest, sugar beet root and sugar yield, sucrose, K+, Na+, and N-amino acid concentrations were not affected by the herbicide treatments compared with those produced in weed-free and herbicide-free plots, indicating that all cultivars managed to overcome the transient pronamide stress. Regarding sugar beet cultivars, root and sugar yield of Avantage and Dorothea at harvest were higher than that of Bianca, whereas sucrose concentration of Avantage was the lowest. There was not an apparent relationship between the order of sugar yield per cultivar (Dorothea > Avantage > Bianca) and the length of the vegetative period (Avantage < Dorothea < Bianca).

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.

Soxhlet extraction and acid digestion methods for silver determination in soils by flame and electrothermal atomic absorption spectrometry

Abstract This paper describes the development of a soxhlet extraction method for the extraction of silver as silver iodide from soil, and its subsequent determination by atomic absorption spectrometry. Silver iodide is extracted as a double salt with potassium iodide in acetone and, after condensation, silver is measured in the extract by flame atomic absorption spectrometry. The efficiency of this extraction was compared to a wet digestion method. Concentrated nitric acid was used for soil digestion and silver was determined in the acid digests by electrothermal atomic absorption spectrometry. In a series of soil samples, silver recovery varied between 90–110%. The detection limits were mass-dependent and, at the experimental conditions described in this work, these were found to be 2 μg/kg in extracted and 3 μg/kg in acid digested soil samples.