Justyna A. Nowakowska

Genetic variability of Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies L. Karst.) natural regeneration compared with their maternal stands

Abstract The genetic variability and biodiversity of tree populations ensure the stability and sustainability of forest ecosystems. New research tools based on molecular DNA markers enable precise characterisation of forest genetic resources, i.e. detection of different allele frequencies in mature trees and progeny populations. The paper describes the genetic structure of mature stands of Scots pine (Pinus sylvestris L.) in Oława Forest District and Norway spruce (Picea abies L. Karst.) in Jawor Forest District and stands of their respective progeny. In the Scots pine stand, there was a slight increase (0.6%) in heterozygosity level and a larger increase (4.9%) in the inbreeding coefficient of progeny trees. In the Norway spruce stand, despite a small reduction (0.9%) in heterozygosity, a similar increase (4.6%) in the inbreeding coefficient of progeny was revealed. In both stands, allele richness and the partition probability of basic clustering were high. Both pine and spruce adults and progeny trees were characterised by high levels of genetic similarity (96% and 79%, respectively). Gene flow between the mature and progeny populations was high (Nm > 1) for both Scots pine and Norway spruce. Conservation of the gene pool within forest tree stands requires an increase in the proportion of natural regeneration. To estimate the extent to which genes are transmitted between adult trees and their progeny, more studies are needed, especially taking into account the influence of silviculture measures, like selective tree cutting, on the genetic variability of the younger generation. These results confirm that the gene pool was conserved when transmitted between the stands studied, as well as highlight the usefulness of such a study for silvicultural purposes

Forest Tree Species Traced with a DNA-Based Proof for Illegal Logging Case in Poland

Precise identification of biological samples remains the most important proof in the forensic science. Illegal logging has become the urgent issue in Poland during the last decades, and conventional methods of investigation turn out to be often insufficient. Recently, the DNA-based markers (SSR and cytoplasmic genes) can remarkably help in the forensic botany performed by the Forest Service Guards and the Police investigation in illegal logging of timber. The identification method relies on comparison of the piece of evidence (i.e., stolen wood fragments) with the piece of reference (e.g., tree parts remained in the forest). We present the usefulness of the DNA neutral markers (i.e., microsatellite loci) and cytoplasmic genes in forensic botany based on several case studies of illegal wood identification in Poland, concerning the most economically important coniferous tree species such as Pinus sylvestris L., Picea abies (L.) Karst., Abies alba Mill., and Larix decidua (L.). Thanks to the DNA profiles established on the basis of minimum 4 microsatellite nuclear DNA loci, and at least one cytoplasmic organelle (mitochondrial or chloroplast) DNA marker, the determination of the DNA profiles provided fast and reliable comparison between material of evidence (also wood and needles) and material of reference (first of all tree stumps) in the forest. These data strongly supported the decision taken by several District Courts in Poland, as far as the identification of wood samples was proved with a high probability (approximately 98–99 %). The aim of the below publication is to present Polish case study on DNA use to fight illegal logging which became very successful among foresters.

Slow Sand Filters as a part of integrated protection of seedlings against disease in forest nurseries

Abstract Slow Sand Filters (SSF) are a biological method used to protect nursery plants, from pathogen infections which can cause serious diseases in many forest tree species. Thanks to SSF application the number of phytopathogens in nurseries can be significantly reduced, as demonstrated by many field and greenhouse experiments (e.g. in Polish nurseries, and for horticultural crops in Germany and The Netherlands). In this study, the effect of pollution from fertilizers and fungicides used in agriculture (e.g. PCNB) on the efficiency of SSFs was assessed. A quantitative analysis was performed of the copiotrophic and oligotrophic bacterial composition colonizing SSF biofilms. The efficiency with which selected Oomycete strains belonging to the genus Phytophthora (P. alni, P. cactorum, P. plurivora) were removed from water was determined based on genetic material (DNA of the organisms) found in the SSF filtrate. Specific primers and TaqMan probes (qPCR) appeared to be the most sensitive molecular methods. Moreover, the microbiological analysis of SSF biofilm performed with selective media allowed the growth of copiothrophic and oligothrophic bacteria to be estimated. The influence of fungicide (PCNB) and N-fertilizer on the number of bacteria in each biofilm was also evaluated. The pollution of water with fertilizer (being used for plant irrigation) was demonstrated to reduce the efficiency of filtration more than fungicide addition (the amount of DNA from those investigated pathogens in the water decreased with time). The amount of bacteria in SSF biofilm readily increased after application of N-fertilizer in contrast to fungicide (PCNB) addition.

Current possibilities and prospects of using fungicides in forestry

Abstract The possibility of using chemicals in European forestry is extremely limited due to the binding legal regulations and specific conditions concerning the market of plant protection products. This is reflected in the limited availability of active fungicides in forestry. Due to this limitation, practitioners using fungicides in forest nurseries and forest cultivation must have substantial knowledge of the biology of pathogens to ensure satisfactorily effective protection. The work presented here provides an overview of the currently recommended fungicides in Polish forestry as well as the mechanisms of interaction between the active substances and the pathogen, the plant and mycorrhizal fungi. The risk of fungicide resistance, which has been insufficiently explored in the context of forest pathogens, is also discussed in this paper.

Analysis of DNA profiles of ash (Fraxinus excelsior L.) to provide evidence of illegal logging

Illegal logging is a major problem in many European countries. Recent progress in molecular biology, however, has significantly improved the ability to accurately identify wood material. In this paper, the first application of microsatellite DNA markers is described in a case of illegal logging of European ash wood in Polish State Forests. The genetic fingerprints of seized ash wood samples were determined using six nuclear and four chloroplast microsatellite loci, characterized by sufficient stringency in forensic analyses. By comparing the DNA profiles obtained, the origin of one sample of ash wood used as evidence material was confirmed, from among three samples serving as reference material with 99.99999% of probability. This work demonstrates how DNA authenticity testing can serve as an important technical tool in monitoring the legality of the suspected ash timber and confirms the utility of these techniques in detecting illegally logged timber in general.

The use of phosphates in forestry

Abstract Phosphite preparations are now an important alternative in plant protection against new, invasive pathogens of the genus Phytophthora and/or Pythium. It is crucial to intervene when alien, invasive oomycetes are carried to plantations or forest stands and attack fine roots via zoospores. The aim of this paper was to demonstrate the possibility of phosphite application to induce resistance to tree pathogens. Phosphate-based fertilizers have been used successfully in nurseries, where application is relatively easy by means of foliar sprays. the traditional fungicides, which are effective in combating fungi, however, fail to control oomycetes. Instead, they mask the disease, which, in turn, causes serious damage to seedlings after they have been planted in a suitable environment. Moreover, the number of effective fungicides available for forest plant protection has continued to decrease in the last decade. The effectiveness of the chemicals is reduced due to their frequent use and their similarity in terms of the active compound or the mechanism of action. Given the low diversity of active compounds, it is necessary to monitor the development of resistance of pathogens to fungicides by means of molecular biology (sequencing and quantitative PCR). Minimising the undesired side effects of chemicals on both, mycorrhizal fungi and pathogens can be achieved by strict adherence to rigorous security measures and, where possible, frequently changing the active compounds to alternatives such as phosphites. The significance of phosphate and phosphite uptake by trees is still a matter of debate, especially under field conditions. Nevertheless, phosphites are environmentally friendly compounds, which constitute an alternative or complement to the traditional chemicals (in accordance with the Directive on Integrated Plant management).

Genetic variability and health of Norway spruce stands in the Regional Directorate of the State Forests in Krosno

Abstract The study was conducted in 2015 in six spruce stands situated in different forest districts administratively belonging to the Regional Directorate of State Forests in Krosno. Each spruce population was represented by 30 trees and assessed in terms of their current health status. Genetic analyses were performed on shoot samples from each tree using nine nuclear DNA markers and one mitochondrial DNA marker (nad1). The health status of the trees was described according to the classification developed by Szczepkowski and Tarasiuk (2005) and the correlation between health classes and the level of genetic variability was computed with STATISTICA (α = 0.05). Nuclear DNA analyses revealed a low level of genetic variability among spruce populations (only 3% of the total genetic variation (FST = 0.028) and a high variability within populations (97%). The total heterozygosity in all stands (HT) was calculated as 0.646. Based on UPGMA analysis, the most genetically similar populations are spruce stands in the Bieszczady National Park and the Ustrzyki Dolne Forest District, which have the smallest genetic divergence of all populations (DN = 0.0165). Our analysis of the mitochondrial gene nad1 revealed the presence of six different haplotypes ‘a’, ‘a1’, ‘b’, ‘c’, ‘d’ and ‘d1’. Comprising 56% of all haplotypes, ‘a’ was the most common showing a predominant impact on spruce migration from the Carpathian area. The analysis based on mitochondrial markers (by Nei) revealed a heterozygosity of 0.525. Based on the observations of disease symptoms, 29% of the trees belong to health class 1, 30% to class 2, 28% to class 3 and class 4 contains 13% of trees. The comparison between health status and the level of genetic variation in the analysed stands showed a positive correlation. Spruce stands with better health were also characterised by a greater degree of genetic variability. Since most of the investigated spruce populations shared the mitochondrial haplotype ‘a’, we have ascertained their Hercynian–Carpathian origin. Only one stand (Cisna) had a high frequency (43.3%) of the Nordic haplotype ‘c’ suggesting that this provenance is derived from the Baltic post-glacial refugium of P. abies in europe.

Application of selected molecular markers in studies on forest trees

Abstract The paper describes a number of molecular methods used in the past and now to analyze forest tree species. Taking into account the economic importance of forest trees and in view of the timber economy, wood properties and characteristics are essential factors subjected to control, observation and research. Molecular techniques that support traditional selection methods allow for genetic diversity analyses considering a range of research aspects. The development of these techniques at the turn of the last two decades has enabled wide-ranging use of molecular data in studies on forest tree populations. On the example of pine (Pinus L.), the paper presents data based on molecular studies as well as a variety of possibilities to apply the obtained results.

Molecular analysis of Phytophthora species found in Poland

Abstract Pathogens of Phytophthora genus are common not only in forest nurseries and stands, but also in water courses. Species of Phytophthora spread with plants for plantings (and soil attached to them) and with water courses as well, attacking the plants growing in riparian ecosystems. Several specialized organisms damaging only one tree species were identified like P. alni on alders or P. quercina on oaks. Some Phytophthora species can develop on several hosts like P. plurivora and P. cactorum on oaks, beeches, alders, ashes and horse chestnuts. Other oomycetes like P. gallica species was found for the first time in Poland in water used for plant watering in forest nursery. Species P. lacustris and P. gonapodyides were found in superficial water. Phytophthora species P. polonica was identified in the declining alder stands for the first time in the world, and P. taxon hungarica and P. megasperma were found in the rhizosphere of seriously damaged ash stands for the first time in Poland. The most often isolated species were P. plurivora (clade 2) with frequency 37% and P. lacustris with frequency 33% (clade 6). The best represented clade 6 revealed the occurrence of 6 species: P. gonapodyides, P. lacustris, P. megasperma, P. sp. raspberry, P. taxon hungarica and P. taxon oak soil.

Four different Phytophthora species that are ableto infect Scots pine seedlings in laboratory conditions

Abstract To investigate susceptibility of young Scots pine seedlings to four Phytophthora species: Phytophthora cactorum, Phytophthora cambivora, Phytophthora plurivora and Phytophthora pini; seven-day-old seedlings of Scots pine (15 seedlings per experiment) were infected using agar plugs of the respective species. Control group also consisted of 15 seedlings and was inoculated with sterile agar plugs. Results unambiguously show that after 4.5 days, all seedlings show clear signs of infection and display severe symptoms of tissue damage and necrosis. Moreover, three and two seedlings in the P. cactorum and P. cambivora infected seedlings groups, respectively, collapsed. The length of largest necrosis measured 13.4±3.90 mm and was caused by P. cactorum. To rule out any putative contamination or infection by secondary pathogens, re-isolations of pathogens from infection sites were performed and were positive in 100% of plated pieces of infected seedlings. All re-isolations were, however, negative in the case of the control group. Detailed microscopic analyses of infected tissues of young seedlings confirmed the presence of numerous Phytophthora species inside and on the surface of infected seedlings. Therefore, our results suggest Phytophthora spp. and mainly P. cactorum and P. cambivora as aggressive pathogens of Scots pine seedlings and highlight a putative involvement of these species in the damping off of young Scots pine seedlings frequently observed in forest nurseries.