Justyna Nowakowska

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 chlo-roplast) 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

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). Minimizing 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).

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.

Concn. of PHO 3 2– ions in plant tissues was detd. by using reaction with Ag + in acidic soln. to metallic Ag ppt. The amt. of Ag was detd. by X-ray fluorescence spectrometry (XRF) and UV-VIS spectrophotometry. The XRF method was less laborious, but its accuracy was lower. Its sensitivity was insufficient to det. very low concns. of phosphonates. The UV-VIS spectrophotometry, after Ag dissoln. in concd. HNO 3 , complexation of Ag + with dithizone, extn. with CHCl 3 and detn. without removal of free dithizone, showed a significantly higher sensitivity. Opracowano metodę ilościowego oznaczania fosfonianów w tkankach roślinnych. Zaproponowana procedura oparta jest na reakcji tych jonów z jonami srebra w środowisku kwaśnym, w wyniku której strąca się osad metaliczne-go srebra. Ilość wolnego srebra, powstałego w wyniku działania fosfonianów, została oznaczona metodami spektrometrii fluorescencji rentgenowskiej XRF i spektrofotometrii UV-VIS. Metoda XRF jest mniej pracochłonna, lecz jej czułość to ok. 0,1 mg srebra, co nie stanowi wyniku zadowalającego w przypadku śladowych ilości fosfonianów w tkankach roślinnych. W przypadku spektrofotometrii UV-VIS, po roztworzeniu srebra w stężonym HNO 3 , skompleksowaniu jonów Ag + ditizonem, ekstrakcji chloroformem i bezpośrednim oznaczeniu spektrofotometrycznym bez usuwania

Oszako T. 2016. Molekularna diagnostyka wybranych patogenów z rodzaju Phytophthora w ramach integrowanej ochrony roślin. Sylwan 160 (5): 365−370. Traditional detection methods such as baiting or direct isolation take a long time and are incapable to handling large volume of material to be tested. The real−time PCR−based techniques are faster, more sensitive, more easily automated, and do not require post−amplification procedures. Species−specific primers for Phytophthora were designed based on the internal transcribed spacer regions (ITS) of rDNA collected from the NCBI DNA database. Primers and probes were designed using the Allele ID 7 at default search criteria. Specific probes were labeled with the reporter dyes JOE (6−carboxy−4,5−dichloro−2,7−dimethoxyfluorescein) at the 5' end and HBQ1 quencher at the 3' end (Sigma−Aldrich). The specificity of primers and fluorogenic probes was tested against genomic DNA of P. alni subsp. multiformis, P. lacustris and P. taxon hungarica. The real−time PCR reactions with the specific probes and primers yielded positive results with five concentrations of standards obtained by standard PCR reaction for corresponding Phytophthora species. The negative control (lack of DNA pathogens) yielded no amplification products. Standard curves showed a linear correlation between input DNA and cycle threshold (Ct) values with R 2 from 0.994 (P. alni) to 0.998 (P. taxon hungarica). The amplification efficiency of target DNA varied from 94.6% (P. alni) to 100% (P. taxon hungarica). The validation of the primers and probes designed for analysed Phytophthora species was performed on pure cultures, on soil samples from the forest nursery and declining oak stands. The designed probes displayed the high specificity of the detection of investigated species in pure cultures. The presented new molecular TaqMan probes can fully assist the integrated pest management as a powerful tool for a quick detection of above pathogenic organisms in forest nurseries. The molecular detection of harmful phytoph− thoras and in consequences diminishing of fungicides use for their control in forestry fully support European Union directives as well as the 'Good plant protection practice measures' elaborated by European and Mediterranean Organisation of Plant Protection.