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Truffle cultivation has quickly grown in European countries and elsewhere, so optimization of the nursery inoculation method to cover the high demand for mycorrhized seedlings is needed. The suitability of two compost-based potting mixes to produce Quercus pubescens Willd. mycorrhized with the black Périgord truffle T. melanosporum Vittad. was tested as an alternative to the employ of traditional potting mix used for mycorrhization and considered as the control treatment. The effects on the mycorrhizal development and the morphometric assessment of the root and shoot system of the downy oak seedlings were investigated 8 months after the spore slurry inoculation in a glasshouse experiment using the root analysis software WinRHIZO®. From the results obtained, the compost mix containing green organic residues from pruning and mowing (Mix 2) achieved better performance with a higher mycorrhization percentage than the control (59.7% and 46.7%, respectively) and significantly higher root growth with enhanced root length (531 cm), number of root tips (3.329), and root forks (3.941) both with respect to the control and the potting mix containing municipal and green organic residues (Mix 1). In conclusion, from this work is evident that potting mix containing recycled organic matter, which is readily available, cheap, and environmentally sustainable can offer excellent mycorrhization performances and may be included in the mycorrhization process of Q. pubescens seedlings with T. melanosporum under controlled conditions.

Ulmus glabra is a deciduous tree with a wide distribution in the Eurosiberian region. The southernmost populations, in the Mediterranean area, are fragmented in mountain areas which act as a refugium. These small relict populations can act as sentinel of global change, including climate change and impacts of human activities such as air pollution. Besides, tropospheric ozone (O3) is an additional stress factor in the Mediterranean region affecting plant physiology and health. Moreover, oxidative stress caused by O3 could increase DNA damage in plants cells. U. glabra 4-year-old seedlings originated from a natural population growing in the Guadarrama mountain range (central Spain), were exposed in Open Top Chambers to four O3 treatments: charcoal filtered air, non-filtered air reproducing ambient levels, non-filtered air supplemented with 15 nl l−1 O3 and non- filtered air supplemented with 30 nl l−1 O3. Ozone effects on the DNA integrity through Comet assay were evaluated and eco-physiological responses were explored as well as. Comet assay showed a significant increase of DNA damage with increasing levels of O3 after only one-month exposure, when no eco-physiological symptoms of damage could be detected. Comet assay could thus be suggested as a predictive test to detect DNA damage induced in plants by other abiotic stresses as well as to identify tolerant and sensitive species or in preservation strategies of small relict populations. The discovery of a test for an early identification of stressed plants could be important to speed the selection of tolerant individuals for breeding programmes.

The data presented in this article are related to the research article titled “Conservation of veteran trees within historical gardens (COVE): a case study applied to Platanus orientalis L. in central Italy” (Ciaffi et al., 2018) [1]. This article reports data on the composition of the substrates used in the different steps of Platanus orientalis micropropagation: establishment of in vitro culture, multiplication, elongation and rooting. Moreover, molecular data were used to assess the genetic fidelity of the micropropagated plants respect mother plants after three year of in vitro cultivation. Fifteen ISSR markers, used in “Determination of genetic stability of long-term micropropagated plantlets of Platanus acerifolia using ISSR markers” (Huang et al., 2009) [2] on P. acerifolia and in “Variant identification in Platanus occidentalis L. using SNP and ISSR markers” (Lee et al., 2012) [3] on P. occidentalis, were successfully employed in the present study on P. orientalis. The plant material was collected from the Renaissance garden of Villa Lante in Viterbo, Italy. It is envisioned that these data set will provide useful information for the conservation of veteran oriental plane trees of historical gardens.

Many veteran trees characterize the landscape of Mediterranean historical parks and gardens, representing unique values from ecological, cultural, social and historical points of view. The absence of natural regeneration is one of the main limits for their conservation. The main purpose of this research was to combine theoretical reasoning and practical solutions to maintain this rich tree heritage. A protocol based on an interdisciplinary approach was developed and applied to the plane trees (Platanus spp.) within the formal garden of Villa Lante of Bagnaia (Viterbo, Italy). The COVE analysis consisted of the following four phases: i) selection of veteran tree species after gathering and comparing historical information; ii) taxonomic identification based on morphological and molecular analyses; iii) tree inventory in GIS environment; and iv) vegetative propagation by woody cuttings and micropropagation. The plane trees surveyed grow on three level terraces and significantly differ in size and phytosanitary conditions according to their topographic position. Molecular analyses indicated a genetic uniformity for the plane trees studied and their memberships to P. orientalis species. This evidence suggests that all specimens belong to the original planting dating back to 1576. All individuals showed a high susceptibility to the pathogen Ceratocystis platani even though so far there is no evidence of canker stain attack. Woody cuttings from the most vigorous individual showed 80% rooting rates. In vitro micropropagation using node culture approach provided healthy plants, maintaining the genetic fidelity respect to mother plant after three years of culture. The obtained results represent a successful example of integrated research for germplasm conservation and management of veteran trees within historical villas in Central Italy.

Global warming is affecting Mediterranean ecosystems, in which Arbutus unedo represents an important species. Mesophyll protoplasts are convenient material to study plant DNA integrity, nevertheless their release from mature plants along the year has not been reported in sclerophyll species. In this respect, in the present study the chance to isolate protoplasts from leaf tissues of A. unedo in different seasons was investigated. The digestion was obtained using Macerozyme R10 (1%) and Cellulase Onozuka RS (2%), with 0.6 M of mannitol, incubated for 4 h at 25 °C in the dark. In spring, soft leaves of different ages were studied to identify the most suitable material and protoplast yields were significantly influenced, with the still expanding 4th leaf characterized by the highest amount. Protoplast release decreased during the growing season when leaves become partially hardened, while their obtainment was quite impossible in summer and winter, due to leaf hardening. In November, an increase of incubation time (16 h) and of Cellulase RS (4%) or mannitol concentration (0.8 M) was needed with chilled leaves from the field; conversely, a good source for protoplasts were the green soft leaves coming from detached and pruned branches placed in controlled environment at spring temperatures. The employment of polyvinylpyrrolidone-40 during isolation procedure is discussed. The isolated protoplasts were employed to assess the DNA integrity, by using single cell gel electrophoresis (SCGE). The X-irradiated nucleoids were significantly damaged confirming that the present approach could be used in the selection of elite material of A. unedo devoted to fruit plantation.

Site conditions and forest management affect dendrometric parameters of chestnut (Castanea sativa Mill.) coppices, but there is modest knowledge on the effect of stand dendrometric characters on physical and mechanical wood characteristics. The aim of this study was to verify these relationships in chestnut coppices that were 12–14 years old. Wood density, compression and bending strength, shrinkages were measured on shoots of five different stand in a vulcanic site in Monte Amiata (Central – Italy). Investigated stands differ in number of stools/ha and dominant height, diameter/basal area of the shoots. The main difference in the physical characters among the stands is density. The initial results of the study showed that physical, mechanical wood characters are more dependent by the shoot than by the site. There is a positive relationships between the number of stools/ha and density and a negative one among shoot dominant height and basal area with wood density. Spectroscopic profile by FTIR has not showed relevant differences among the stands. Wood anatomy has showed the breakpoint at cellular level.

Mediterranean countries are expected to experience a notable increase in average air temperatures and an alteration of precipitation patterns, distribution, intensity and duration, as a result of global climate changes. In this region, wetlands and coastal areas are increasingly at risk, as they are particularly exposed to a range of hazards connected to climate change, such as drought, flooding, and soil salinity. Nevertheless, the species which inhabit these areas are likely to be well adapted to future conditions caused by global warming effects. Among them, Tamarix species have been reported to be highly tolerant to many abiotic stresses including salinity, drought, flooding, and extreme temperatures. Exploring their diversity could be fundamental, as genotypes can be selected for their natural tolerance to some particular stress, and may be conserved and used in restoration practices under the perspectives of global climate changes. Moreover, recent experimental evidence supported the employment of Tamarix spp. as bio-fuel crops. There are eleven Tamarix species in Italy, occupying coastal dunes and the riverbanks of Southern regions. The most widespread species are Tamarix gallica and Tamarix africana. Although they play a fundamental ecological role in dunes’ fixation and in inhabiting salinized areas, which would otherwise be subjected to desertification, they are still not well known. In this study, we introduce Italian Tamarix spp. ecological, physiological, morphological and genetic diversity with the aim of creating awareness as regards their potential use for the recovery of degraded areas in the Mediterranean Basin.

Tamarix L. plants are tolerant to extreme environmental conditions and represent a resource for the recovery of marginal areas. The aim of this study is to develop a molecular method for species assignment and to characterize the genetic differentiation of Italian Tamarix populations. Blind sampling was performed and individuals were gathered without any regard for species identity from seven sites in Italy. If possible, flowers for species identification were collected, but 60% of samples remained unidentified. The genotypic profile of 17 microsatellite markers and a Bayesian statistical approach allowed the individuals to split among genetic entities rather than by their species identity. A clear assignment of Tamarix africana Poir. individuals was found, but this was not the case for Tamarix gallica L. and Tamarix canariensis Willd., whose individuals were clustered in a unique group (T. gallica-like). In T. africana, the Bayesian analysis of the genetic structure pointed out the existence of a unique gene pool, whereas according to principal coordinates analysis (PCOA) and FSTvalues, the populations from Lazio and Sardinia were more differentiated. All the analyses performed showed a differentiation between Sicily and peninsular Southern Italy in the T. gallica-like group. This study is the first to report the characterization of the natural genetic resources of Italian tamarisks and it suggests the absence of genetic differentiation between T. gallica and T. canariensis.

Tamarix L. is a taxonomically difficult genus since species identification is based on hard to see floral characters, which can be observed only during the very short spring bloom. In order to find new and always accessible tools for species identification, leaf epidermis characters were analyzed. During spring 2008, flowers and green twigs were collected in four natural tamarisk populations of Southern Italy. The flowers were used for species identification, while leaf imprints were examined for stomata and salt glands traits. Tamarix africana Poir. and Tamarix gallica L. were the most widespread species in the studied populations. T. africana showed longer guard cells, and lower stomatal and salt gland density compared to T. gallica. These differences were more significant than intraspecific ones. Moreover, the interspecific leaf micromorphological differentiation was found to be consistent across the populations. These results suggest the efficacy of the use of leaf morphological traits as a new tool f...

In this study, the genes of the Aux/IAA family were used as functional markers to characterise bud break stages in a white poplar clone. In the first experiment, under greenhouse conditions, the sprouting of repressed sylleptic buds was obtained by pruning the shoot tip during growing season. Buds were collected at 0, 6, 24 and 48 h after pruning for molecular analyses. A decrease in transcript level of IAA4 and IAA8 genes was observed in the first and second bud below the cut after 6 and 24 h, respectively. In the second experiment, bud break of post-dormant proleptic buds was induced by forcing in climatic chamber. The first 6 and the following 6 buds below the apical one were sampled every 48 h during forcing. Anatomical studies were also carried out on buds and plants were equipped with stem and bud radial growth sensors to check their swelling. In both experiments, gene expression patterns showed a decrease and a successive increase in expression of IAA4 and IAA8 genes during bud break. The transient down-regulation of these genes was observed only in buds that formed new branches. Thus, similar molecular mechanisms are involved in bud break of both sylleptic and proleptic buds.

White poplar (Populus alba L.) is native to Eurasia and is unexploited for its growth potential and stress-adaptive mechanisms. A better knowledge of its genome will allow for more effective protection and use of critical genetic resources. The main objective of this study was the construction of highly informative P. alba genetic maps. Two genotypes were selected from contrasting natural Italian populations and crossed to generate an F1 mapping pedigree. Amplified fragment length polymorphism and simple sequence repeat markers were used to genotype 141 F1 individuals. The pseudo-testcross strategy was applied for linkage analysis. The generated maps showed good overall colinearity to each other and allowed for a complete alignment with the 19 haploid chromosomes of the Populus genome sequence. The locus that determines sex as a morphological trait was positioned on a non-terminal position of LG XIX of the female parent map. Comparison among Populus species revealed differences in the location of the sex locus on LG XIX as well as inconsistencies in the heterogametic sex. The genetic analysis of the sex locus in P. alba provides insights into sex determination in the genus and is useful for the identification of sex-linked markers and the early assessment of plant gender. Furthermore, these genetic maps will greatly facilitate the study of the genomics of Populus and how it can be exploited in applied breeding programs.

The effect of salt stress on leaf morphology and functionality was studied in three Populus alba genotypes differing in tolerance to salinity: 6K3 (sensitive), 2AS11 (moderately tolerant), and 14P11 (tolerant). Plants were subjected to an intense and progressive salt stress from 50 to 250 mM NaCl by 50 mM steps at 10-day intervals. The micromorphological results highlighted phenotypic variation among the three genotypes already in control plants, with the genotype 14P11 having significantly smaller epidermal cells and higher stomatal density. Salt-treated plants modulated differently the expansion of stomata compared with epidermal cells. Regression analysis showed significant correlations between decrease of stomatal area and stomatal conductance (gs) in genotypes 14P11 and 6K3. So, the common reduction of stomatal area could be an early mechanism to save water in this species. However, only genotype 14P11 showed further significant decrease of this trait under the highest salinity level, combined with a significant reduction in leaf length. In addition, this genotype showed the lowest leaf abscission rate at the end of salt stress period. The genotype 6K3 was severely affected by leaf necrosis and showed the highest leaf abscission rate in salt stress conditions. In the moderately tolerant genotype 2AS11, an intermediate plastic behaviour in both leaf morphology and physiology was observed during the experiment. The phenotypic variation among the three genotypes in terms of micromorphology and stomatal conductance is discussed in relation to plant functionality in salt stress conditions. Overall results suggest that leaf morphological habit contributes to salt tolerance in P. alba.

Quercus ilex L. seedlings were exposed in open-top chambers for one growing season to three levels of ozone (O3 ): charcoal filtered air, non-filtered air supplemented with +30% or +74% ambient air O3 . Key functional parameters related to photosynthetic performance and stomatal density were measured to evaluate the response mechanisms of Q. ilex to chronic O3 exposure, clarifying how ecophysiological traits are modulated during the season in an ozone-enriched environment. Dark respiration showed an early response to O3 exposure, increasing approximately 45% relative to charcoal-filtered air in both O3 enriched treatments. However, at the end of the growing season, maximum rate of assimilation (Amax ) and stomatal conductance (gs ) showed a decline (-13% and -36%, for Amax and gs , respectively) only in plants under higher O3 levels. Photosystem I functionality supported the capacity of Q. ilex to cope with oxidative stress by adjusting the energy flow partitioning inside the photosystems. The response to O3 was also characterised by increased stomatal density in both O3 enriched treatments relative to controls. Our results suggest that in order to improve the reliability of metrics for O3 risk assessment, the seasonal changes in the response of gs and photosynthetic machinery to O3 stress should be considered.

The functional response to salt stress was studied in three Populus alba L. genotypes, from three localities of Italy: genotype 6K3 from northern Italy, genotype 2AS11 from middle-southern Italy, and genotype 14P11 from a coastal zone in southern Italy. Plants of the three genotypes were submitted to a progressive salt stress by NaCl irrigation during 4 weeks. The severity of symptoms was linked to the sodium accumulated in leaves of salt-stressed plants. Genotype 2AS11 accumulated significantly less sodium in leaves than the other genotypes, arising the hypothesis that mechanisms for sodium exclusion at root level could exist in this genotype. Its height growth was significantly reduced by salt stress but diameter growth and leaf abscission were not affected. 14P11 and 6K3 accumulated significantly more sodium in leaves and they were significantly affected by leaf abscission and reduction of height and diameter growth. Genotype 6K3 was the most salt-sensitive and its leaves had more extensive necrosis than 14P11 and 2AS11. The transcription profile of plasma membrane H + -ATPase and tonoplast H + -ATPase genes revealed a different regulation in response to salt stress in 2AS11 and 14P11, but it was unchanged in 6K3. The different response to salinity could be related to different ability to exclude sodium in roots and to different regulation of ion transport across membranes of leaf cells. The differences observed could be the expression of evolutive adaptation to the ecological conditions of original provenances. Likely a different genetic basis underlies the different degree of salt tolerance observed.

Summary • The effects of elevated CO 2 on leaf development in three genotypes of Populus were investigated during canopy closure, following exposure to elevated CO 2 over 3 yr using free-air enrichment. • Leaf quality was altered such that nitrogen concentration per unit d. wt ( N mass ) declined on average by 22 and 13% for sun and shade leaves, respectively, in elevated CO 2 . There was little evidence that this was the result of ‘dilution’ following accumulation of nonstructural carbohydrates. Most likely, this was the result of increased leaf thickness. Specific leaf area declined in elevated CO 2 on average by 29 and 5% for sun and shade leaves, respectively. • Autumnal senescence was delayed in elevated CO 2 with a 10% increase in the number of days at which 50% leaf loss occurred in elevated as compared with ambient CO 2 . • These data suggest that changes in leaf quality may be predicted following longterm acclimation of fast-growing forest trees to elevated CO 2 , and that canopy longevity may increase, with important implications for forest productivity.

Unlike herbaceous, annual crops, trees are not highly domesticated and, therefore, have wild relatives with which they are interfertile. They are also long-lived perennials that produce copious amounts of pollen and seed, which are often disseminated over considerable distances by the wind. Federal regulators have made it clear that before transgenic trees can be grown commercially in the U.S., it will be necessary to develop a strategy to mitigate the risk of transgene spread into the environment. One way to satisfy this requirement is to genetically engineer reproductive sterility. Because of its many useful attributes, poplar has becomethe model tree species for research community. However, because of its relatively long juvenile period, the development of a reliable sterility system for poplar is taking longer than expected. By having an early-flowering genotype of poplar, it will be possible to make much faster progress in our efforts to develop a reliable transgene-confinement system. We have identified a genotype ofPopulus alba that can be induced to flower within nine months of being regenerated.

Soil salinity is an important limiting factor to tree growth and productivity. Populus alba L. is a moderately salt-tolerant species and its natural populations are adapted to contrasting environments, thus providing genetic resources to identify key genes for tolerance to abiotic stress, such as salinity. To elucidate the molecular and genetic basis of variation for salinity tolerance in P. alba, we analyzed the short-term ecophysiological and transcriptome response to salinity. Two contrasting genotypes, 6K3, salt sensitive, and 14P11, salt tolerant, originating from North and South Italy, respectively, were challenged with salt stress (200 mM NaCl). Sodium accumulated in the leaves of salt-treated plants and its concentration increased with time. The net photosynthesis was strongly reduced by salinity in both genotypes, with 6K3 being significantly more affected than 14P11. The transcriptional changes in leaves were analyzed using cDNA microarrays containing about 7000 stress-related poplar expressed sequence tags (EST). A microarray experiment based on RNA pooling showed a number of salinity--regulated transcripts that markedly increased from 3 h to 3 days of salinity treatment. Thus, a detailed analysis was performed on replicated plants collected at 3 days, when ~20% of transcripts showed significant change induced by salinity. In 6K3, there were more genes with decreased expression than genes with increased expression, whereas such a difference was not found in 14P11. Most transcripts with decreased expression were shared between the two genotypes, whereas transcripts with increased expression were mostly regulated in a genotype-specific manner. The commonly decreased transcripts (71 genes) were functionally related to carbohydrate metabolism, energy metabolism and photosynthesis. These biological processes were consistent with the strong inhibition of photosynthesis, caused by salinity. The commonly increased transcripts (13 genes) were functionally related to primary metabolism and biosynthesis of proteins and macromolecules. The salinity-increased transcripts discriminated the molecular response of the two genotypes. In 14P11, the 21 genes specifically salinity-induced were related to stress response, cell development, cell death and catabolism. In 6K3, the 15 genes with salinity-increased expression were involved in protein biosynthesis, metabolism of macromolecules and cell organization and biogenesis. The difference in transcriptome response between the two genotypes could address the molecular basis of intra-specific variation of salinity tolerance in P. alba.

Tamarix plants are resistant to abiotic stresses and have become invasive in North America. Their taxonomy is troublesome, and few molecular makers are available to enable species identification or to track the spread of specific invasive genotypes. Transcriptome sequencing projects offer a potential source for the development of new markers. •Thirteen polymorphic simple sequence repeats (SSRs) markers derived from Expressed Sequence Tags (ESTs) from Tamarix hispida, T. androssowii, T. ramosissima, and T. albiflonum were identified and screened on 24 samples of T. africana to detect polymorphism. The number of alleles per locus ranged from two to eight, with an average of 4.3 alleles per locus, and the mean expected heterozygosity was 0.453. •Amplification products of these 13 loci were also generated for T. gallica. These new EST-SSR markers will be useful in genetic characterization of Tamarix, as additional tools for taxonomic clarification, and for studying invasive populations where they are a threat.

The wide development of single nucleotide polymorphism (SNP) markers also in non-model species increases the need for inexpensive methods that do not require sophisticated equipment and time for optimization. This work presents a new method for polymerase chain reaction (PCR) amplification of multiple specific alleles (PAMSA), which allows efficient discrimination of SNP polymorphisms in one reaction tube with standard PCR conditions. This improved PAMSA requires only three unlabeled primers: a common reverse primer and two allele-specific primers having a tail of different length to differentiate the two SNP alleles by the size of amplification products on agarose gel. A destabilizing mismatch within the five bases of the 3? end is also added to improve the allele specificity. To validate the accuracy of this method, 94 full-sib individuals were genotyped with three SNPs and compared to the genotypes obtained by cleaved amplified polymorphic sequence (CAPS) or derived CAPS. This method is flexible, inexpensive, and well suited for high throughput and automated genotyping. © 2007 Springer-Verlag.

Leaf expansion in the fast-growing tree,Populus × euramericana was stimulated by elevated [CO2] in a closed-canopy forest plantation, exposed using a free air CO2 enrichment technique enabling long-term experimentation in field conditions. The effects of elevated [CO2] over time were characterized and related to the leaf plastochron index (LPI), and showed that leaf expansion was stimulated at very early (LPI, 0–3) and late (LPI, 6–8) stages in development. Early and late effects of elevated [CO2] were largely the result of increased cell expansion and increased cell production, respectively. Spatial effects of elevated [CO2] were also marked and increased final leaf size resulted from an effect on leaf area, but not leaf length, demonstrating changed leaf shape in response to [CO2]. Leaves exhibited a basipetal gradient of leaf development, investigated by defining seven interveinal areas, with growth ceasing first at the leaf tip. Interestingly, and in contrast to other reports, no spatial differences in epidermal cell size were apparent across the lamina, whereas a clear basipetal gradient in cell production rate was found. These data suggest that the rate and timing of cell production was more important in determining leaf shape, given the constant cell size across the leaf lamina. The effect of elevated [CO2] imposed on this developmental gradient suggested that leaf cell production continued longer in elevated [CO2] and that basal increases in cell production rate were also more important than altered cell expansion for increased final leaf size and altered leaf shape in elevated [CO2].

Water relations of three Eucalyptus species (E. x trabutii Wilm., E. viminalis Labill., E. dalrympleana Maid.), widely planted in the Mediterranean basin, were analyzed throughout an entire year in relation to natural cold hardening. Osmotic potential, both at saturation and at the turgor loss point, showed a greater reduction during hardening in the more frost-resistant E. viminalis and E. dalrympleana than in the more frost-sensitive E. x trabutii. The hardening capabilities of all species were analyzed in relation to the freezing dehydration index, FDI, a parameter derived from pressure-volume analysis which represents the water lost when cells, initially at the turgor loss point, attain thermodynamic equilibrium with extraplasmatic ice. The FDI at the killing temperature showed little variation either between frost-sensitive and frost-resistant species, or between hardened and non-hardened plants. The index may, therefore, be useful for evaluating a plant's potential for injury by freeze-induced desiccation.

Eucalyptus plantations of the Mediterranean countries are frequently jeopardized by the occurrence of relatively severe frosts every 5–10 years. In order to examine the mechanisms of frost injury in the species generally utilized in the region, whole seedlings and samples of tree tissues of different species (Eucalyptus occidentalis Endl., E. × trabutii Vilm., E. globulus Labill., E. gunnii Hook. f.) were tested by artificial frost regimes. Non-hardened vs. cold-hardened plants, woody vs. leaf tissues and stem pieces of different sizes were compared for their supercooling ability, exotherm characteristics and frost tolerance. The species most often utilized for planting in the Mediterranean region, E. × trabutii, E. occidentalis and E. globulus did not show any capability to respond to artificial or natural hardening regimes; however, the first two species were irreversibly damaged as soon as ice formed in the tissues, probably because of ‘non-equilibrium freezing’. Eucalyptus globulus showed a modest capability to withstand extracellular ice and, therefore, ‘equilibrium freezing’. Eucalyptus gunnii, a subalpine species utilized for interspecific crosses, was able to respond to hardening pretreatments and to withstand, in the hardened state, extracellular ice formation and temperatures as low as −14°C.