Marilena Idžojtić

Leaflet morphometric variation of service tree (Sorbus domestica L.) in the Balkan Peninsula

Abstract In most European countries, the service tree (Sorbus domestica L.) is a rare and threatened species and its conservation has been recognised as a priority. The aim of this study was to asses its morphologic variation in the western and central part of the Balkan Peninsula and in southern Central Europe. Three populations were analysed: one in Serbia, one in Bosnia and Herzegovina and one in Slovenia. In each population 30 trees were selected, and from each tree 30 leaves were collected for morphometric analysis based on nine leaflet morphological traits. Univariate (ANOVA) and multivariate (MANOVA) analysis of variance were used to estimate the variation within- and between populations and a discriminant analysis was performed to examine the structure of the between-population differences. The values of particular morphological traits found in our study did not differ considerably from the values reported elsewhere. The results revealed significant within- and between population variation. Variation within populations was highly significant for all the scored leaf morphological traits, while variation between populations was significant for all the studied traits except for the leaflet length. The discrimination between the three populations was significant. High percentages of correctly classified samples demonstrate good discriminating employability of the analysed leaf morphological traits and indicate differentiation of the analysed populations.

Castanea sativa: genotype-dependent recovery from chestnut blight

In Lovran (coastal Croatia), a unique forest/orchard of evenly mixed grafted marrons and naturally growing nongrafted sweet chestnut trees exists. This old chestnut population has been devastated by chestnut blight, caused by an aggressive introduced pathogenic fungus, Cryphonectria parasitica. However, initial observations indicated recovery of naturally growing chestnut trees in that area, mediated by Cryphonectria-associated hypovirus (Cryphonectria hypovirus 1 (CHV-1)). Such recovery was not observed on grafted trees. Genotyping both, we confirmed the clonal origin of the grafted ones—marrons. No significant difference was observed between fungal strains isolated from naturally growing trees and the ones from marrons regarding fungal vegetative compatibility types or the prevalence of CHV-1. A strong correlation was observed between the types of canker: active/deep-expanding versus healing callus or superficial necrosis and the absence or presence of CHV-1 in the fungal isolates, sampled from naturally growing trees (Spearman rho 0.686, p value 7.81 × 10−5, Kendall tau 0.686, p value 5.18 × 10−7). Such correlation was not observed on marrons (Spearman rho 0.236, p value 0.235, Kendall tau 0.236, p value 0.084), because, unexpectedly, active/deep-expanding cankers were often associated with hypovirulent fungal isolates. These data indicate that the lack or unequal distribution of naturally occurring hypovirulence were not the cause of substantial marron decay in Lovran. Ecological and age-dependant differences were ruled out because all sampled trees are growing in close proximity and are of similar age. The results imply that the marron genotype is especially vulnerable and its ability to recover is limited even when the hypovirulent strain of the fungus is present in the canker.

The incidence of mistletoe (Viscum album ssp. abietis) on silver fir (Abies alba) in Croatia

The research on incidence of mistletoe (Viscum album ssp. abietis) on silver fir (Abies alba) was carried out in natural fir stands in Croatia. In the area of Gorski Kotar 32.8 % of the examined dominant fir trees were infected with mistletoe. The mistletoe incidence was presented according to the damage degrees of silver fir from 0 (healthy trees) to 4 (dead trees), rated by the crown defoliation and needle discoloration. With the increase of incidence, mistletoe spreads more onto more vital, i.e. less damaged trees. In the Dinaric Alps 28.6% of the examined fir trees were infected, this percentage amounting to 27.1% for the mountainous regions between the Sava and Drava rivers.The site and stand parameters (exposure, elevation, site quality, forest community and crown closure) were analysed in order to establish whether there was a correlation between these parameters and mistletoe incidence. There was a negative correlation between the elevation and mistletoe incidence. Among the compartments with a closed stands there were considerably less compartments with higher mistletoe incidence than among compartments with a sparsely closed and understocked stands. The other analysed site and stand parameters individually had no significant influence on mistletoe incidence.The correlation of silver fir mortality in 2004 and mistletoe incidence in 2002/03 was analysed, and their strong correlation was established. Mistletoe could be considered as a bioindicator of silver fir decline, and probably a significant contributor to that decline. In the areas where mistletoe incidence is great it can be presumed that silver fir is significantly damaged.

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.

Morphologic variation in northern marginal Juniperus oxycedrus L. subsp. oxycedrus populations in Istria

Marginal tree populations are believed to be more differentiated and host less variation than central tree populations. The aim of this study was to perform a detailed morphometric study of J. oxycedrus L. subsp. oxycedrus in northern marginal populations in order to establish its phenotypic variation and geographical differentiation and to examine the eventual presence of putative species J. deltoides in Istria. Morphologic variation was studied in 16 northern marginal populations in Istria. Nine morphological traits from a minimum of 50 leaves from each of 206 individuals and two morphological traits from 30 to 50 seed cones in each of 103 females were measured. Phenotypic variation in Istrian populations was high and of a similar magnitude to that found in the centre of the distribution area. Gender dimorphism in leaf morphology was detected, but the study failed to confirm its uniform distribution pattern. Significant and surprisingly high among-population differentiation (8–30%) was revealed. Average leaf shape in the study area is clearly “delta-shaped” characterised by a relatively wide leaf base. However, no significant differences in any of the studied traits were found between the marginal Istrian populations and the more central populations analysed in our earlier studies.

‘Candidatus Phytoplasma pini’ in pine species in Croatia

Seven conifer species: Cupressus sempervirens, Juniperus communis, J. oxycedrus, Picea abies, Pinus halepensis, P. mugo and P. nigra were tested for the presence of phytoplasma DNA. Using nested PCR (primer pairs P1/P7 followed by R16F2n/R16R2), ‘Candidatus Phytoplasma pini’ was detected in five out of 10 sampled P. halepensis trees and in one out of 11 sampled P. mugo trees. Symptoms like yellowing, proliferations and shedding of the needles were observed on sampled P. halepensis trees, while yellowing and dieback of shoots and branches were observed on P. mugo. Phytoplasma positive P. halepensis trees were found along the Adriatic coast, whereas the only positive P. mugo was found approximately 50 km inland. All ‘Ca. P. pini’ isolates obtained from conifers in Croatia had the same 16S rDNA sequence. The sequence obtained from the amplified fragments of 16S rDNA had a 100% level of identity with the 16S rDNA sequences of ‘Ca. P. pini’ previously found in Spain, Poland and Czech Republic.

Alive and kicking, or, living on borrowed time? – Microsatellite diversity in natural populations of the endangered Ulmus minor Mill. sensu latissimo from Croatia

Abstract The main objective of this research was to assess the genetic diversity of 5 natural field elm populations in Croatia. The study results suggest that the observed populations are characterized by a satisfactory amount of heterozygosity, and that the impact of the Dutch elm disease on the amount of genetic diversity in the sampled populations is currently negligible. However, one population displayed a significant excess of heterozygosity, implying a genetic bottleneck. The existence of a very clear genetic differentiation between the continental and the Mediterranean populations of Ulmus minor in Croatia was noticed.

Hosts and distribution of Viscum album L. ssp. album in Croatia and Slovenia

Abstract Viscum album L. ssp. album is semi-parasitic on deciduous trees and shrubs. In order to identify hosts and map the distribution of V. album ssp. album in Croatia and Slovenia, field research was carried out, and herbaria were surveyed. In Croatia and Slovenia, V. album ssp. album occurred on 59 taxa. In Croatia, there were 52 hosts (33 autochthonous and 15 allochthonous species, two cultivars and two hybrids). In Slovenia, there were 25 hosts (21 autochthonous and four allochthonous species). There were 18 hosts common to both countries, 34 hosts were found only in Croatia, and seven hosts only in Slovenia. The hosts belonged to 13 families. The majority of these (19 species) belong to the Rosaceae, followed by Salicaceae, Aceraceae, Betulaceae, Fagaceae, Juglandaceae, Tiliaceae, Hippocastanaceae, Ulmaceae, Oleaceae, Fabaceae, Moraceae and Viscaceae. All hosts have been previously recorded in the literature, except Alnus japonica (Thunb.) Steud., Amelanchier lamarckii F.G. Schroed. and Crataegus nigra Waldst. et Kit. The distribution of this mistletoe was scattered, due to the scattered distribution of hosts, local conditions, movement of bird-vectors, etc. A continuous distribution was found only in part of the distribution area of narrow-leaved ash (Fraxinus angustifolia Vahl).

Changes in Cryphonectria parasitica populations affects 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.

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.