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This paper reports the results of the study on the new type of necrotic lesion observed on epicormic shoots and leaf petioles of Fraxinus excelsior showing ash decline symptoms in stands of the Miechow Forest District, southern Poland. The performed symptom analysis included: shape and size of necroses, discoloration of necrotic tissue and the occurrence of fungal fructification. The same species of fungus, which was initially identified as Boeremia exigua, was isolated from 97% of epicormic shoots and from 82% of leaf petioles. However, detailed morphological and physiological (NaOH test) analyses and combined ITS, ACT, TUB and EF1-α phylogeny proved these isolates to belong to Boeremia lilacis, the species known so far as Syringa vulgaris pathogen occurring occasionally also on Forsythia hybrids or Philadelphus. Fraxinus excelsior is reported for the first time as a host for this fungus. To clarify the possible role of B. lilacis in development of necrotic lesions on ash, 10 randomly selected isolates were tested for their pathogenicity toward young F. excelsior plants using the method of artificial wound inoculation under field conditions. The isolates used in the test caused tissue necroses on all 60 inoculated shoots and all 60 inoculated leaf petioles. Necrotic lesions developed as a result of artificial inoculation corresponded largely to the symptoms following natural infections.

Common beech is a shade-tolerant tree species that can adapt to varying light intensities at the level of whole plants, crown fragments, and individual leaves. The acclimation abilities of the morphological, physiological, and biochemical characteristics of beech leaves were experimentally determined in tree crowns for different levels of light availability. About 24% higher length, width, and area and about 35% higher thickness were recorded in the sun leaves compared with shade leaves. Lower and earlier maximum leaf pigment levels, a faster degradation of leaf pigments during senescence, and a higher chlorophyll a/b ratio were observed in fully sun-grown leaves compared with leaves growing deeper in the canopy. Changes in the intensity of oxidative stress and the differential ability of developing and senescing leaves to defend against this effect under different light conditions were determined. This resulted in a higher redox imbalance and faster senescence in the outer parts of the tree crowns. Due to higher ascorbic acid and glutathione content and slower activity loss of antioxidative enzymes involved in superoxide anion and hydrogen peroxide decomposition, better control over the redox balance, oxidative stress, and senescence induction was noted in the sun leaves.

High-altitude tree populations are exposed to severe natural environmental conditions. Among abiotic factors, variable temperatures, early frosts, and high radiation are the factors affecting tree growth at high altitudes. Fagus sylvatica L. exhibits a variety of physiological and genetic traits that allow it to adapt to different forest habitats. This study examines the differences in the biochemical properties of senescing beech leaves between populations originating from different altitudes using a common-garden experiment. Leaves were collected from five-year-old plants from the beginning of August to the end of October for two years. Based on the changes in senescence marker levels the genetic differences and significant correlations between populations’ altitude origin and their biochemical characteristics were identified. According to the free radical theory of leaf senescence, reactive oxygen species (ROS) and senescence markers were highly correlated. In this study, populations from higher altitudes were characterized by earlier and greater increases in ROS content and oxidative stress, which resulted in higher antioxidative system activity. Increases in ROS in high-altitude populations play a controlling role to initiate earlier senescence processes that allow the trees to adapt to harsh climatic conditions. Earlier senescence allows beech trees to maintain a balance between nitrogen metabolism and photosynthetic activity. It allows for remobilization of nitrogen compounds more efficiently and protects the trees from nitrogen loss and prepares them for winter dormancy.

High levels of heavy metals can lead to multiple toxic effects in plants. They have capability to influence plant growth and development by direct inhibition of molecular, biochemical, and physiological processes or through the production of reactive oxygen species (ROS), which can inhibit most cellular processes at various levels of metabolism. Plants counteract the heavy metal oxidative stress by inducing plant scavenging system including overexpressing of numerous signaling and antioxidative proteins and nonenzymatic antioxidants. Different efficiencies of ROS scavenging system during various environmental stress including heavy metals can result in developing plants that can overcome oxidative stress. The capability of plant species to accumulate and tolerate heavy metals has a great importance for plant selection useful for phytoremediation. This article details the toxicity of heavy metals, the sites of production, and the role of ROS as oxidative damage inducers and the link between heavy metals, oxidative stress, and antioxidative system activity in different plant species. The type of phytoremediation methods and mechanisms of plant tolerance for heavy metal toxicity are also presented.

The aim of the study was to investigate the effect of soil recovery after a landslide on the nutritional status of a young generation of Silver fir (Abies alba Mill.) and Silver birch (Betula pendula Roth.) trees. The study was carried out on the site of a landslide that took place in 2010 in the Gorlice Forest District (Southern Poland). Basic soil properties, macro, and microelements content and enzymatic activity were determined in the soil samples that were collected from across the landslide area, from locations distributed by a grid 10 &times, 10 m (18 points). Plant material samples, collected to indicate nutritional status, were also taken from each point. Results demonstrate that the nutritional status of emerging regeneration depends on soil acidification and soil organic matter content. The pH of the soils on the landslide determines the intensity of nutrient uptake by the young seedlings. The nutrition of young trees varied across the landslide zone, differing in terms of the soil&rsquo, s organic matter content and its chemical properties. In comparison to the depletion zone, the accumulation zone proved to be substantially richer in soil organic matter, resulting in higher soil biochemical activity. The study demonstrates that Silver birch (Betula pendula Roth.) is improving nutrient cycling in areas disturbed by landslide.

Girdling was applied to 5-year-old potted beech individuals of early, intermediate and late phenological forms to block assimilate export from leaves. Phloem severance caused accumulation of soluble carbohydrates and starch in leaves and increased the C/N ratio. While the hexose content increased continuously until the end of the experiment, the sucrose and starch contents peaked earlier, depending on the plant’s phenological features. Different rates of chlorophyll degradation and H2O2and TBARS (thiobarbituric acid-reactive substances) production in different phenological forms implied that phloem girdling was the source of oxidative stress and, depending on the phenological form, accelerated leaf senescence to different degrees. The variable rate of the increase in soluble carbohydrate and starch content, characteristic of the different phenological forms, had different modifying effects on the antioxidant activity in leaves. Compared with the early phenological form, the late form was characterized by a smaller increase in H2O2and TBARS content and delayed and slowed chlorophyll and carotenoid degradation. In conjunction with the larger increase in the activity of antioxidant enzymes (catalase, ascorbate peroxidase and superoxide dismutase) induced by carbohydrate accumulation and slower carotenoid degradation, these changes led to the late form having greater resistance to oxidative stress and slower senescence.

A large part of the area in Europe in which Fraxinus excelsior is native is currently affected by ash dieback, a threatening disease caused by the ascomycetous fungus Hymenoscyphus fraxineus. Fungi other than H. fraxineus also occur in large numbers on stems of the dying ash trees. To clarify their possible role in the dieback process, six fungal species common on dying stems and twigs of ash in Poland, i.e. Cytospora pruinosa, Diaporthe eres, Diplodia mutila, Fusarium avenaceum, F. lateritium and F. solani, were tested for pathogenicity using a test based on artificial wound inoculations of 6-year-old F. excelsior plants under field conditions, with H. fraxineus included for comparison. There were significant differences in index of pathogenicity among the fungi tested. Hymenoscyphus fraxineus (mean index 5.78) was the most pathogenic. Diplodia mutila (4.23) and C. pruinosa (4.02) were significantly less pathogenic than H. fraxineus, but significantly more than the other fungi. Diaporthe eres (2.43), F. avenaceum (1.92), F. solani (1.86) and F. lateritium (1.08) were the least pathogenic (P

F. excelsior is affected by dieback in the major part of its natural geographical range in Europe, which results in economic and ecological losses. The disease is caused by the ascomycetous fungus Hymenoscyphus fraxineus, a pathogen introduced to Europe most probably from East Asia. This paper presents data on fungi identified on F. excelsior trees representing two different stages of ash dieback in Poland. Fungal communities were identified in initial necrotic lesions on living stems and twigs using the classical method of isolation on malt extract agar and morphological and molecular analyses. In dead apical parts of stems and twigs, fungi were identified by microscopic analyses of fruit bodies formed in situ. Seventy-one fungal taxa were found in 720 samples with symptoms of initial or advanced necrosis. The most common fungus detected in initial necrotic lesions in each forest site was Hymenoscyphus fraxineus (59.2 % of analysed samples). Other frequently isolated fungi included Alternaria alternata, Diaporthe eres, Diplodia mutila, Fusarium avenaceum, F. lateritium and Phomopsis spp. Fruit bodies on dead apical parts of stems and twigs were produced mostly by Diaporthe eres, Diplodia mutila, Lophiostoma corticola, Phomopsis spp., Sirodothis sp. and Valsa cypri. Fungal communities from different sites were similar, as shown by high Sorensen similarity index values. Greatest variation in fungal community structure at the initial necrotic stage was realized by D. mutila and F. avenaceum, and at the advanced necrotic stage by D. eres, D. mutila and Phomopsis spp. Data show a close affinity of certain fungi to site, suggesting geographical relatedness. The ecological effects of distribution of the invasive H. fraxineus and of other fungi sporulating on diseased ash trees in Europe are discussed.

Plant tissue culture techniques have become an integral part of progress in plant science research due to the opportunity offered for close study of detailed plant development with applications in food production through crop improvement, secondary metabolites production and conservation of species. Because the techniques involve growing plants under controlled conditions different from their natural outdoor environment, the plants need adjustments in physiology, anatomy and metabolism for successfulin vitropropagation. Therefore, the protocol has to be optimized for a given species or genotype due to the variability in physiological and growth requirement. Developing the protocol is hampered by several physiological and developmental aberrations in the anatomy and physiology of the plantlets, attributed toin vitroculture conditions of high humidity, low light levels and hetero- or mixotrophic conditions. Some of the culture-induced anomalies become genetic, and the phenotype is inherited by clonal progenies while others are temporary and can be corrected at a later stage of protocol development through changes in anatomy, physiology and metabolism. The success of protocols relies on the transfer of plantlets to field conditions which has been achieved with many species through stages of acclimatization, while with others it remains a challenging task. This review discusses various adjustments in nutrition, physiology and anatomy of micro-propagated plants and field grown ones, as well as anomalies induced by thein vitroculture conditions.

The frost resistance of one year old needles and shoots of Abies grandis (seven provenances) and Abies alba (one provenance) was determined using Dexter's method since early autumn till late spring (September-May). Material was collected from trees over ten year old, planted in Beskid Sądecki Range near Krynica, 800 m above sea level. In this study it was found, that in early autumn (September) A. grandis develops weaker frost hardiness than Abies alba. In the late autumn and in winter, provenances coming from region II (according to Müller), and especially provenance 12016, are equal in respect to frost hardiness of shoots and needles to Abies alba. Among the provenances from Müller's region I exceptional frost hardiness is displayed by provenance 12005, which is also characterized by fast growth. In spring, Abies alba flushes earlier than A. grandis, which makes it more susceptible to late frosts.

□ The effect of controlled mycorrhization of pine seedlings withH. crustuliniforme and L. bicolor on seasonal variation and content of photosynthetic pigments, carbohydrates, and proteins was investigated. Non-inoculated seedlings and seedlings inoculated with H. crustuliniforme contained more chlorophylls and carotenoids than seedlings inoculated with L. bicolor. Seedlings inoculated with L. bicolor showed slower accumulation of soluble carbohydrates than inoculated with H. crustuliniforme. During the whole vegetation period, the insoluble carbohydrates content was higher in seedlings inoculated with L. bicolor than with H. crustuliniforme and non-inoculated plants. Plants inoculated with H. crustuliniforme contained a higher amount of soluble proteins than non-inoculated and inoculated with L. bicolor plants. A time-course analysis showed that non-inoculated plants contained more insoluble proteins only at the beginning of the vegetation period. Plants inoculated with H. crustuliniforme contained more i...

Reactive oxygen species affect the timing and rate of leaf senescence in different phenological forms of beech. We investigated the contribution of the enzymatic part of the antioxidative system in controlling the induction and course of this process. Leaf senescence was associated with differential dynamics of protein degradation and increased accumulation of the superoxide ion (O 2 ·− ) and hydrogen peroxide (H2O2). The different phenological forms and temperature courses over the sampling periods significantly affected the level and profile of antioxidative enzyme activity. Except for catalase (CAT), the activity of antioxidative enzymes (superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) in the pre-senescence stage was highest in the late form. Diverse responses to decreased temperature caused differential rates of decrease in antioxidative enzyme activity. While CAT and MDHAR activity decrease was continuous, characteristic of phenological forms and showed a time shifting, the decrease in SOD, APX, and GR activity was reversible during the temperature increase in the initial stage of senescence and dependent on phenological form. In the final stage of senescence, enzyme activity decrease was quick, was irreversible, and was less influenced by temperature. Changes in antioxidative enzyme activity resulted in greater tolerance to oxidative stress and lower rates of leaf senescence in the late phenological form. Based on current and previous studies, the hypothetical model for slower leaf senescence in late phenological forms of beech was demonstrated.

Eighty-three strains of Sclerophoma pythiophila were isolated in the period between 1996 and 2006 from needles and shoots of Pinus sylvestris displaying various types of disease symptoms or damages caused by Contarinia baeri or Thecodiplosis brachyntera. On the basis of fifty-six RAMS markers, very high genetic variability of examined strains was ascertained (mean value of Jaccard's coefficient 0.58). The highest genetic similarity was shown by strains related with needles damaged by Contarinia baeri (0.65), whereas the lowest by those derived from dead shoot tips (0.53). No monomorphic markers were found for individual groups of strains, yet on the basis of Nei's genetic distance matrix, it was possible to determine a group of closely related fungus populations which was connected with the damaging of the needles by C. baeri or T. brachyntera (Nei's coefficient ranging from 0.035 to 0.059) and populations related with the occurrence of necrosis on shoots or decay of their tips (Nei's coefficient - 0.066). The PCA confirmed genetic similarity of strains related with damaging of the needles by insects and strains isolated from local necroses on shoots and withered shoot tips. A high level of genetic variability between populations was shown by AMOVA analysis. A high level (14.9%) and statistically significant (P=0.001) share of between-population genetic variability were ascertained.

On the basis of morphological features and RAPD markers the strains of Chalara ovoidea found in Poland on planks and on stems of beech trees were identified. As reference strains the cultures taken from CBS Utrecht were employed; they were cultures CBS 354.76 and CBS 136.88. The amplification of genomic DNA was conducted using 10 primers (OPA01-OPA10), 7 of which (OPA01-OPA05, OPA09, OPA10) gave positive results. In total 42 fragment of DNA (bands) were obtained. In case of primers OPA03, OPA04, OPA05, and OPA09 all obtained fragments for analyzed strains were fully monomorphic. This means, that no genetic variability was found using the above mentioned primers. Low genetic variability was ascertained in the analysis of frequency of occurrence of DNA fragments using other primers, namely OPA01, OPA02, and OPA10. The matrix and dendrogram of genetic affinities among different strains of Chalara, calculated using the Jaccard’s similarity coefficient suggested, that the most similar strains are the ones coming from Poland (HMIPC 16136 and HMIPC16664) as well as the strain CBS 136.88, while somewhat different from them is the strain CBS 354.76. To determine, how exactly did the dendrogram reflect genetic affinity among analyzed strains, the Mantel’s test was employed. The correlation coefficient amounted to 0.78, suggesting that the strains under study had been grouped properly. The results showed, that the fungal strains found in southern Poland represent the species Chalara ovoidea.

A total of 159 colonies of Chalara fraxinea were isolated between 2005 and 2006 from dying trees of European ash (Fraxinus excelsior L.) aged between 3 and 10 years. They derived from five regions of Poland differing by geographic location and climatic conditions. On the basis of 90 RAMS markers, pathogen intra- and inter-population variability, as well as its dependency on geographic distance and climatic conditions in the regions of strain origin, was analysed. The applied measures of intrapopulation genetic variability (genetic distance, Nei’s unbiased diversity, Shannon’s Information Index and percentage of polymorphic loci) allowed for differentiation of two strain groups: the first deriving from lowlands and the second from uplands and mountainous areas. Strains in lowlands were characterised by smaller number of markers, smaller number of polymorphic loci and smaller intrapopulation genetic variability. Positive and statistically significant correlation was shown between variability of isolates and elevation of regions above sea level. Pair-wise genetic distances between groups of isolates (Nei’s unbiased genetic distance) from particular regions were not significantly correlated with the corresponding geographic distances. On the basis of AMOVA, it was shown that 85% of variability was within-region differences and 2% between-region differences, whereas differences between lowlands and uplands were 13%. Principal Components Analysis (PCA) for the investigated regions confirmed the results from Nei’s genetic distance matrix.

Between 1996 and 2006, 97 strains of Sclerophoma pythiophila were isolated from 3 to 10-year-old trees of Pinus sylvestris from three regions of Poland differing by climatic conditions and geographic location. On the basis of 56 random amplified microsatellites (RAMS) markers obtained with the use of five primers, very high level of genetic variability of strains (mean Jaccard's coefficient 0.56) was ascertained. Among the analysed markers only one was completely monomorphic, whereas six were monomorphic in 90%. Greater genetic similarity was ascertained for strains in southern Poland (0.58) in comparison with strains deriving from northern Poland (0.52). Correlation between the percentage share of polymorphic loci and the average annual temperature of places of strain isolation (r = 0.62) were shown, as well as the number of days with snow cover (r = 0.79). On the basis of the AMOVA analysis, it was ascertained that 11.6% of genetic variability was located between regions of the strains origin and 88.4% within the regions.

Summary Thirty-nine strains of Gremmeniella abietina were isolated between 1980 and 2005 from diseased or symptomless Pinus sylvestris and Pinus nigra, or sporadically Pinus jeffreyi and Pinus armandii from four regions of Poland with varying climatic conditions. Thirty-five of the strains were genetically similar to the G. abietina A type found in Scandinavia, whereas four were similar to the B type. On the basis of random amplified microsatellite markers, very high intrapopulation and interpopulation genetic variation was detected among the strains. The average value of the Jaccard genetic similarity coefficients among strains was 0.56 for A type and 0.47 for B type, and the monomorphic loci partition was small, at 14% of all analysed loci for the A type. The degree of genetic variation of the fungal strains within each of the four regions was similar, showing no significant differences. The Jaccard coefficient for the strains isolated from P. sylvestris and P. nigra differed significantly, at 0.57 and 0.54 respectively. The genetic distance among A type strains from the investigated regions, expressed using Nei’s coefficient, was not correlated with geographic distance; however, it was highly dependent on elevation (correlation coefficient r = 0.92).

This study examines changes in the reactive oxygen species (ROS) content and in the level and degree of ascorbate and glutathione oxidation during leaf senescence in phenological forms of beech (Fagus sylvatica L.). Leaves were collected between August and October in 2009 and 2012. Chlorophyll degradation depended significantly on the phenological characteristics of the specimens. Chlorophyll, proteins, superoxide ion and hydrogen peroxide contents and lipid peroxidation dynamics exhibited greater dependence on temperature changes in the early form than in the late form. In the early stage of senescence, the late beech form presented a greater increase in ascorbate and glutathione synthesis than did the early form (ascorbate and glutathione synthesis was the main characteristic of the phenological forms’ defence mechanism against increasing ROS concentration). The early form was characterized by a stronger correlation between increasing ROS and the concentrations of oxidized forms of ascorbate and glutathione. We conclude that the earlier onset and greater rate of autumn senescence in the early phenological form relative to the late form were associated with significantly quicker chlorophyll degradation and greater production of ROS, which was better balanced by the antioxidant system in the late phenological form. The different concentrations of ascorbate and glutathione and the degree of their oxidation influenced the oxidative stress tolerance in the senescing leaves of the different phenological forms of beech.

Summary The distribution of genetic diversity in a local population of Dothistroma septosporum was determined on a microspatial geographical scale. The study was conducted in a seed orchard of Pinus nigra divided into four plots, each of 0.84 ha in area. Ninety-two isolates were obtained from needles with red band needle blight symptoms. Molecular variance, Mantel test and autocorrelation spatial analyses were performed with the use of random amplified microsatellites markers to test the null hypothesis of a random distribution of genotypes. The groups of isolates from individual plots demonstrated small differences as regards intrapopulation variation. The mean contribution of polymorphic loci for isolate groups was almost 77%, genetic diversity 0.270, while the Shannon coefficient was 0.389. The Mantel test demonstrated a significant, positive correlation between Huff's genetic distance and geographical distance. Significant spatial genetic structure was detected with autocorrelation coefficients being significant in the first two distance classes up to about 8–12 m.

(Received 28 March 2008; accepted 25 November 2008) Abstract • Microsatellite markers were used to describe the genetic structure and variability of early, interme- diate and late phenological forms of European beech (Fagus sylvatica L.). Two hundred and seventy individuals from three populations located in southern Poland were divided into three forms according to the phenological criterion - bud burst, and analyzed for allelic variation at five highly polymorphic microsatellite loci. • Population differentiation was moderate and differed significantly among phenological forms. Av- erage values of FST and RST decreased across phenological forms and amounted to FST values of 0.135, 0.110 and 0.108 and RST values of 0.365, 0.231 and 0.098 for early, intermediate and late forms of beech, respectively. • Analysis of Molecular Variance (AMOVA) revealed different genetic structures characteristic of respective phenological forms of beech. The amount of within-population variability increased with the delay of the beginning of vegetation and amounted to 64%, 77% and 90% of total variability, depending on phenological form. A similar trend was found in average pairwise genetic distance be- tween individuals belonging to a given phenological form (11.78, 11.85 and 12.22, from early to late forms). • Our results demonstrate the importance of late spring frosts as a factor influencing the genetic struc- ture of beech, and as a cause of the decrease in genetic variability as well as the increase in population differentiation proportional to the degree of phenological earliness.

Investigations were conducted on the strongly infested by microbes embryos isolated from seed stratified in non-sterile conditions or from tree buds. The results showed, that PPM (Plant Preservative Mixture, produced by Plant Cell Technology, Inc.) was very useful for increasing the percentage of sterile cultures disinfected in a 5% solution of NaOCl. The best way of applying PPM turned out to be adding the substance directly to the growing medium. At the concentration of 2 or 4 cm3 x dm-3 of PPM there was an 30% increase in percentage of sterile of in vitro cultures developed from embryos and 70-80% of cultures developed from buds, as compared with the control without PPM. At the range of concentrations employed in this study, no negative effects of PPM upon the development and growth of callus were observed.