Patterns of Leaf and Fruit Morphological Variation in Marginal Populations of Acer tataricum L. subsp. tataricum.

Marginal populations are usually smaller and more isolated and grow in less favourable conditions than those at the distribution centre. The variability of these populations is of high importance, as it can support the adaptations needed for the conditions that they grow in. In this research, the morphological variability of eight Tatar maple (Acer tataricum L. subsp. tataricum) populations was analysed. Tatar maple is an insect-pollinated and wind-dispersed shrub/tree, whose northwestern distribution edge is in southeastern Europe. Morphometric methods were used to analyse the variability of the populations using leaf and fruit morphology. The research revealed significant differences between and within populations. Furthermore, differences in the distribution of the total variability were noted, which suggest that different evolutionarily factors affect different plant traits. Correlation analysis confirmed a weak dependency between the vegetative and generative traits. In addition, no evidence was found for the presence of isolation by environment (IBE). However, the Mantel test for isolation by distance (IBD) was significant for the leaf morphometric traits and non-significant for the fruit morphometric traits. Being the marginal leading-edge populations, they are younger and were less likely to have had time for adaptation to local environments, which would have resulted in the development of IBE. Overall, edge populations of Tatar maple were characterised by great morphological variability, which helps these populations in their response to the intensive selective pressures they face in their environment.

Physiological variations in hypovirus-infected wild and model long-term laboratory strains of Cryphonectria parasitica.

Introduction: Forest ecosystems are highly threatened by the simultaneous effects of climate change and invasive pathogens. Chestnut blight, caused by the invasive phytopathogenic fungus Cryphonectria parasitica, has caused severe damage to European chestnut groves and catastrophic dieback of American chestnut in North America. Within Europe, the impacts of the fungus are widely mitigated through biological control that utilizes the RNA mycovirus: Cryphonectria hypovirus 1 (CHV1). Viral infections, similarly to abiotic factors, can cause oxidative stress in their hosts leading to physiological attrition through stimulating ROS (reactive oxygen species) and NOx production. Methods: To fully understand the interactions leading to the biocontrol of chestnut blight, it is vital to determine oxidative stress damage arising during CHV1 infection, especially considering that other abiotic factors, like long-term cultivation of model fungal strains, can also impact oxidative stress. Our study compared CHV1-infected C. parasitica isolates from two Croatian wild populations with CHV1-infected model strains (EP713, Euro7 and CR23) that have experienced long-term laboratory cultivation. Results and Discussion: We determined the level of oxidative stress in the samples by measuring stress enzymes' activity and oxidative stress biomarkers. Furthermore, for the wild populations, we studied the activity of fungal laccases, expression of the laccase gene lac1, and a possible effect of CHV1 intra-host diversity on the observed biochemical responses. Relative to the wild isolates, the long-term model strains had lower enzymatic activities of superoxide dismutase (SOD) and glutathione S-transferase (GST), and higher content of malondialdehyde (MDA) and total non-protein thiols. This indicated generally higher oxidative stress, likely arising from their decades-long history of subculturing and freeze-thaw cycles. When comparing the two wild populations, differences between them in stress resilience and levels of oxidative stress were also observed, as evident from the different MDA content. The intra-host genetic diversity of the CHV1 had no discernible effect on the stress levels of the virus-infected fungal cultures. Our research indicated that an important determinant modulating both lac1 expression and laccase enzyme activity is intrinsic to the fungus itself, possibly related to the vc type of the fungus, i.e., vegetative incompatibility genotype.

Gene flow between wild trees and cultivated varieties shapes the genetic structure of sweet chestnut (Castanea sativa Mill.) populations.

Gene flow between cultivated and wild gene pools is common in the contact zone between agricultural lands and natural habitats and can be used to study the development of adaptations and selection of novel varieties. This is likely the case in the northern Adriatic region, where centuries-old cultivated orchards of sweet chestnut (Castanea sativa Mill.) are planted within the natural distribution area of the species. Thus, we investigated the population structure of several orchards of sweet chestnuts. Furthermore, the genetic background of three toponymous clonal varieties was explored. Six genomic simple sequence repeat (gSSR) and nine EST-derived SSR (EST-SSR) loci were utilized in this research, and both grafted and non-grafted individuals were included in this study. Five closely related clones were identified, which represent a singular, polyclonal marron variety, found in all three cultivation areas. Furthermore, many hybrids, a result of breeding between cultivated and wild chestnuts, have been found. Analyzed semi-wild orchards defined by a diverse genetic structure, represent a hotspot for further selection and could result in creation of locally adapted, high-yielding varieties.

Phenotypic Variation in European Wild Pear (Pyrus pyraster (L.) Burgsd.) Populations in the North-Western Part of the Balkan Peninsula.

Leaves play a central role in plant fitness, allowing efficient light capture, gas exchange and thermoregulation, ensuring optimal growing conditions for the plant. Phenotypic variability in leaf shape and size has been linked to environmental heterogeneity and habitat characteristics. Therefore, the study of foliar morphology in plant populations can help us to identify the environmental factors that may have influenced the process of species diversification. In this study, we used European wild pear (Pyrus pyraster (L.) Burgsd., Rosaceae) as a model species to investigate the phenotypic variability of leaves under different environmental conditions. Using leaf morphometric data from 19 natural populations from the north-western part of the Balkan Peninsula, a high level of variability among and within populations were found. Leaf traits related to leaf size were more variable compared to leaf shape traits, with both influenced by geographic and environmental factors. Consequently, patterns of isolation by environment (IBE) and distance (IBD) were identified, with IBE showing a stronger influence on leaf variability. Multivariate statistical analysis revealed that European wild pear populations from the north-western part of the Balkan Peninsula can be divided into two morphological clusters, consistent with their geographical distance and environmental conditions. Our results confirm a high level of phenotypic variability in European wild pear populations, providing additional data on this poorly studied species, emphasizing phenotypic plasticity as a major driver in the adaptation of this noble hardwood species to rapid climate change.

Morphological and Chemical Diversity and Antioxidant Capacity of the Service Tree (Sorbus domestica L.) Fruits from Two Eco-Geographical Regions.

Service tree, Sorbus domestica L., is a rare and neglected wild fruit tree species of southern and central Europe. Being distributed in different eco-geographical regions, with fragmented and low-density populations, S. domestica represents an interesting model case for investigating patterns of within- and between-population diversity at geographical and environmental scales. This study aimed to analyze the proximate composition, antioxidant activity, and morphometric fruit characteristics. We examined the diversity and population divergences of 49 S. domestica individuals originating from seven populations across continental and Mediterranean eco-geographical regions. In addition, tests of isolation by distance and environment were performed to detect the magnitude of divergence explained by geographic and environmental variables. Significant differences between the studied populations were found in almost all of the studied morphometric and chemical fruit characteristics. The studied service tree populations were characterized by high phenotypic variation despite the low number of trees per population. Model-based population structure analysis using morphometric and chemical fruit characteristics revealed three groups of service tree populations. We concluded that non-effective pollen and seed dispersal along with genetic drift and specific environmental factors resulted in a distinct phenotype with a specific chemical composition in the isolated island population. In addition, a pattern of isolation by the environment was revealed. We infer that morphological and chemical differences between the studied populations in the true service tree from different eco-geographical regions were mediated by adaptation to the specific environmental conditions.

Temporal and Spatial Genetic Population Structure of Cryphonectria parasitica and Its Associated Hypovirus Across an Invasive Range of Chestnut Blight in Europe.

Chestnut blight has spread throughout Europe since the introduction of its causal agent, Cryphonectria parasitica, >70 years ago. In our study, we analyzed the diversity of vegetative compatibility (vc) and microsatellite genotypes of C. parasitica, as well as sequence diversity of Cryphonectria hypovirus 1 (CHV1) in six populations from Switzerland, Croatia, and North Macedonia. Resampling of local populations that were already investigated more than a decade ago allowed us to analyze the spatial and temporal population structure across an invasive range of the pathogen in Europe. Regardless of which genetic marker was used, the >60-year-old Swiss and Croatian populations had high population diversity, whereas more recent North Macedonian populations were mostly clonal. These diversity differences between the investigated populations remained stable over time. A high diversity of CHV1 was observed in all three countries, with North Macedonian strains forming a separate cluster from strains obtained in other countries. No correlation between vc diversity and CHV1 prevalence was observed, suggesting a well-established and maintained natural hypovirulence in all countries, further corroborated by an observed increase in genetic diversity of Croatian C. parasitica populations over time, without collapse of CHV1 prevalence.

Influence of F1 hybridization on the metal uptake behaviour of pine trees (Pinus nigra x Pinus thunbergiana; Pinus thunbergiana x Pinus nigra).

Pine needles have been considered to be useful bio-indicators for air pollution. This phenomenon can be used for environmental studies for monitoring purposes. Additionally, this fact offers the possibility to study uptake and accumulation behaviour not only in different species, but also in hybrids obtained from common pine trees to inheritage processes. Therefore, needles of Pinus nigra Arnold and Pinus thunbergiana Franco as well as of their F1 hybrids were investigated for essential and non-essential metals, such as Al, B, Ba, Ca, Cd, Co, Cu, Cr, Fe, K, Mg, Mn, Mo, Na, Ni, Pb, Sr, and Zn. The samples underwent acidic microwave-assisted digestion prior to analysis inductively coupled plasma atomic emission spectrometry (ICP-AES). Furthermore flavonoids were quantitatively determined to prove hybrid character. Regarding all determined analytes, increase and decrease of uptake in the needles of the hybrids were evaluated in comparison to the needles of the parent pine species to see which parent is the dominant one. In the hybrids higher amounts of Al, Cd, Mo, Ni, Mg, Mn, and Zn were found. Different behaviour was registered for flavonoids than for metals, due to different metabolic pathways.

Changes in Cryphonectria parasitica Populations Affect Natural Biological Control of Chestnut Blight.

Invasive species, especially plant pathogens, have a potential to completely eradicate native plant species and remodel landscapes. Tripartite interactions among sweet chestnut (Castanea sativa), chestnut blight-causing invasive fungus Cryphonectria parasitica, and hyperparasitic virus Cryphonectria hypovirus 1 (CHV1) were studied in two populations. The number of different vegetative compatibility (vc) types of C. parasitica more than doubled over the 10 years, while the hypovirulence incidence dropped in one population and slightly increased in the other one. Over the course of our 3-year monitoring experiment, the prevalence of hypovirulent isolates obtained from monitored cankers increased slowly (i.e., more hypovirulent isolates were being obtained from the same cankers over time). Within studied cankers, considerable changes in vc type and CHV1 presence were observed, indicating a highly dynamic system in which virulent and hypovirulent mycelia, sometimes of discordant vc types, often appeared together. The increase in hypovirulence prevalence did not have any observable curative effect on the cankers and, occasionally, reactivation of healed cankers by new, virulent C. parasitica isolates was observed. Both short- and long-term observations and revalidation of the infected plant populations are necessary to accurately estimate disease progress and formulate an adequate disease management strategy.

Biological control of chestnut blight in Croatia: an interaction between host sweet chestnut, its pathogen Cryphonectria parasitica and the biocontrol agent Cryphonectria hypovirus 1.

BACKGROUND: Chestnut blight, caused by the fungus Cryphonectria parasitica, is a severe chestnut disease that can be controlled with naturally occurring hypoviruses in many areas of Europe. The aim of this research was to measure the effect of different Cryphonectria hypovirus 1 (CHV1) strains on the growth of the fungal host and select strains that could potentially be used for human-mediated biocontrol in forests and orchards, and to investigate whether and how chestnut-fungus-virus interactions affect the development and growth of the lesion area on cut stems. RESULTS: Two Croatian CHV1 strains (CR23 and M56/1) were selected as potential biocontrol agents. The sequencing of CHV1/ORF-A showed that both of these virus strains belonged to the Italian subtype of CHV1. In vitro transfection of selected virus strains from hypovirulent to genetically diverse virus-free fungal isolates and subsequent inoculation of all virus/fungus combinations on stems of genetically diverse sweet chestnut trees revealed that Croatian virus strain CR23 had an equally hypovirulent effect on the host as the strong French strain CHV1-EP713, while M56/1 had a weaker effect. Furthermore, it was shown that in some cases the same hypovirus/fungus combinations induced various degrees of canker development on different chestnut genotypes. CONCLUSION: Some CHV1 strains belonging to the Italian subtype have similar hypovirulent effects on C. parasitica to those belonging to the French subtype. Furthermore, chestnut susceptibility and recovery could be influenced by the response of chestnut trees to particular hypovirulent C. parasitica isolates, and virus-fungus-chestnut interactions could have significant implications for the success of chestnut blight biocontrol. © 2016 Society of Chemical Industry.

Morphological Characterization and Chemical Compositionof Fruits of the Traditional Croatian Chestnut Variety'Lovran Marron'.

'Lovran Marron' is the only known traditional Croatian variety of the sweet chestnut. The objective of this study is to specify qualitative and quantitative morphological characteristics and to analyze the chemical composition of the 'Lovran Marron' fruits as well as to compare them to Marusnjak fruits (trees from the 'Lovran Marron' seed) and fruits from the local natural sweet chestnut population. Seven morphological characteristics were measured: fruit mass, height, width and thickness, scar length and width, and the length of the longest intrusion of the seed coat into the kernel. Eight qualitative characteristics were estimated: embryony, degree of penetration of the seed coat into the kernel, fruit shape, glossiness and colour, kernel colour, hairiness towards the top of the fruit and the existence of longitudinal stripes. 'Lovran Marron' has desirable qualitative and quantitative fruit characteristics. The Marusnjak trees mostly have intermediate morphological traits of fruits in comparison with 'Lovran Marron' and trees from the natural population. 'Lovran Marron' had the smallest mass fractions on average of: K, Mg, Ca, Na, Mn, Cu and Fe. The highest mass fractions on average of these macro- and microelements were characteristic of the trees from the natural population. The highest average content of carbohydrates was recorded in the 'Lovran Marron' and the lowest in the fruits from the natural population. The Marusnjak fruits had intermediate water, protein, ash, carbohydrates, and macro- and microelement content. The content of Cd and Pb was lower in all samples than the maximum allowed amounts in the sweet chestnut fruits.

High molecular diversity in the true service tree (Sorbus domestica) despite rareness: data from Europe with special reference to the Austrian occurrence.

BACKGROUND AND AIMS: Sorbus domestica (Rosaceae) is one of the rarest deciduous tree species in Europe and is characterized by a scattered distribution. To date, no large-scale geographic studies on population genetics have been carried out. Therefore, the aims of this study were to infer levels of molecular diversity across the major part of the European distribution of S. domestica and to determine its population differentiation and structure. In addition, spatial genetic structure was examined together with the patterns of historic and recent gene flow between two adjacent populations. METHODS: Leaf or cambium samples were collected from 17 populations covering major parts of the European native range from north-west France to south-east Bulgaria. Seven nuclear microsatellites and one chloroplast minisatellite were examined and analysed using a variety of methods. KEY RESULTS: Allelic richness was unexpectedly high for both markers within populations (mean per locus: 3·868 for nSSR and 1·647 for chloroplast minisatellite). Moreover, there was no evidence of inbreeding (mean Fis = -0·047). The Italian Peninsula was characterized as a geographic region with comparatively high genetic diversity for both genomes. Overall population differentiation was moderate (FST = 0·138) and it was clear that populations formed three groups in Europe, namely France, Mediterranean/Balkan and Austria. Historic gene flow between two local Austrian populations was high and asymmetric, while recent gene flow seemed to be disrupted. CONCLUSIONS: It is concluded that molecular mechanisms such as self-incompatibility and high gene flow distances are responsible for the observed level of allelic richness as well as for population differentiation. However, human influence could have contributed to the present genetic pattern, especially in the Mediterranean region. Comparison of historic and recent gene flow may mirror the progress of habitat fragmentation in eastern Austria.