The winter survival of the invasive pathogen Phytophthora alni subsp. alni in black alder stems was studied in the bankside alder stand of the Moravská Dyje River in southern Bohemia after two very different winter seasons: cold in 2008/2009, with the average temperature of -1.96°C, and extremely mild in 2006/2007, with the average temperature of 2.54°C. The difference in these two winters resembles the expected potential climate change in Central Europe in this century. After the cold winter of 2008/2009, the pathogen survived in only 13.91% of the samples, with the average survival rate of 2.70%. The pathogen survived the mild winter much better and was successfully isolated from 86.09% of the samples, with the average survival rate of 25.52%. Moreover, the total thickness of the covering tissues (outer + inner bark) and exposure to the most heated southwestern quadrant of stem girth positively affected the pathogen survival. Winter freezing seems to be an important environmental factor regulating the pathogen survival in alder stems and disease severity.
In pathogenic fungi and oomycetes, interspecific hybridization may lead to the formation of new species having a greater impact on natural ecosystems than the parental species. From the early 1990s, a severe alder (Alnus spp.) decline due to an unknown Phytophthora species was observed in several European countries. Genetic analyses revealed that the disease was caused by the triploid hybrid P. × alni, which originated in Europe from the hybridization of P. uniformis and P. × multiformis. Here, we investigated the population structure of P. × alni (158 isolates) and P. uniformis (85 isolates) in several European countries using microsatellite markers. Our analyses confirmed the genetic structure previously observed in other European populations, with P. uniformis populations consisting of at most two multilocus genotypes (MLGs) and P. × alni populations dominated by MLG Pxa-1. The genetic structure of P. × alni populations in the Czech Republic, Hungary and Sweden seemed to reflect the physical isolation of river systems. Most rare P. × alni MLGs showed a loss of heterozygosity (LOH) at one or a few microsatellite loci compared with other MLGs. This LOH may allow a stabilization within the P. × alni genome or a rapid adaptation to stress situations. Alternatively, alleles may be lost because of random genetic drift in small, isolated populations, with no effect on fitness of P. × alni. Additional studies would be necessary to confirm these patterns of population diversification and to better understand the factors driving it.
In the Czech Republic, Phytophthora alni was first confirmed in 2001 and the pathogen has been quickly spreading and occupying almost the whole area of the country. The pathogen attacks Alnus glutinosa or A. incana to a lesser extent and causes considerable losses of alder trees along hundreds of kilometres of riverbanks. The aim of our work was to perform the identification of P. alni isolates at the subspecific level using PCR and to determine the frequencies and distribution of particular subspecies. The allele-specific PCR primers focused on allele diversity of orthologs of ASF-like, TRP1, RAS-Ypt, and GPA1 genes were selected for identification. Eighty-eight per cent of the 59 analysed isolates belonged to P. alni ssp. alni while 12% were P. alni ssp. uniformis. P. alni ssp. multiformis has not been recorded in the country till now. The two subspecies differed in distribution. P. alni ssp. alni dominated in riparian stands along broader rivers in lowlands and the results confirmed the more effective spreading of P. alni ssp. alni based on its higher aggressiveness and ecological advantage. P. alni ssp. uniformis was acquired rather from riparian stands of small watercourses at higher altitudes. The insular distribution of P. alni ssp. uniformis may represent the remains of its former occurrence. Therefore, P. alni ssp. uniformis may be an indigenous subspecies suppressed by the more aggressive related taxon.
Summary The impact of ash dieback caused by Hymenoscyphus fraxineus on 17 provenances of Fraxinus excelsior and one provenance of Fraxinus angustifolia was studied in an extensive field trial established in the Czech Republic prior to the H. fraxineus invasion in 1999. A difference in the level of resistance to ash dieback between the species was found: F. angustifolia was significantly less affected by the disease than F. excelsior . Moreover, particular provenances of F. excelsior showed important differences in the level of resistance to H. fraxineus . A relationship between the impact of ash dieback and altitude was also discovered – the provenances from altitudes above 600 m a.s.l. were less affected by the pathogen than were the provenances from lower areas. No difference in the impact of the disease among provenances of F. excelsior from different ecotopes (ravine, calcareous ravine and alluvial) was found. Substantial among‐tree variability in resistance to H. fraxineus was observed throughout the trial – promising genotypes (with crown defoliation up to 5%) were identified in all 18 tested provenances. In regard to this finding, it appears that the main source of resistance to the pathogen is probably at the individual genotype level in the trial. A secondary but massive attack by Hylesinus fraxini was identified in the trees that had been greatly damaged by ash dieback, and the beetle caused their health to deteriorate significantly. A significant negative effect of the presence of collar necroses caused by H. fraxineus and browse damage was also identified.
Summary Twenty‐six commercial formulations of fungicides at six concentrations were evaluated in vitro for their efficacy on mycelial growth of Hymenoscyphus fraxineus (anamorph Chalara fraxinea ). The results are presented as EC 50 , EC 90 and minimal inhibitory concentration values; the comparisons with the recommended application concentrations showed that 10 of the 26 fungicides were highly effective in their ability to inhibit the mycelial growth of the pathogen. The eight most effective fungicides identified based on multiple comparisons analysis were azoxystrobin, bitertanol, captan (in two‐component preparation with trifloxystrobin), difenoconazole, kresoxim‐methyl, spiroxamine (in multicomponent preparation with tebuconazole and triadimenol), tebuconazole and trifloxystrobin. Azoxystrobin, difenoconazole, kresoxim‐methyl, mancozeb, myclobutanil, pyrimethanil, tebuconazole and trifloxystrobin were selected to verify their effectiveness in in planta tests. During two field tests in nurseries, it was found out that tebuconazole (triazole), trifloxystrobin and kresoxim‐methyl (strobilurins) and mancozeb (dithiocarbamate) were significantly effective against H. fraxineus . The best results were gained with tebuconazole, when mean percentage of diseased saplings was 16.3%, whereas in the control, it was 63.6%. The combination and alternation of fungicides from the triazole, strobilurine and dithiocarbamate chemical groups should be an effective tool for protecting ash saplings in forest nurseries. Moreover, exact timing of the treatment reflecting rainfall and development of first symptoms in the foliage will be also important.
The number of described species of the oomycete genus Phytophthora is growing rapidly, highlighting the need for low-cost, rapid tools for species identification.Here, a collection of 24 Phytophthora species (42 samples) from natural as well as anthropogenic habitats were genetically identified using the internal transcribed spacer (ITS) and cytochrome c oxidase subunit I (COI) regions.Because genetic identification is time consuming, we have created a complementary method based on by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).Both methods were compared and hypothesis that the MALDI TOF method can be a fast and reliable method for the identification of oomycetes was confirmed.Over 3500 mass spectra were acquired, manually reviewed for quality control, and consolidated into a single reference library using the Bruker MALDI Biotyper platform.Finally, a database containing 144 main spectra (MSPs) was created and published in repository.The method presented in this study will facilitate the use of MALDI-TOF MS as a complement to existing approaches for fast, reliable identification of Phytophthora isolates.
Bleeding canker and decline of riparian alder populations has been an important problem in many European countries, including the Czech Republic. Initially, an 'alder Phytophthora' was isolated from damaged black alder trees in western Bohemia near the town of Karlovy Vary in 2001 (Černýet al., 2003). The exact species identification was unknown at this time. Since its original discovery, we have isolated similar Phytophthora species from damaged innerbark and conductive tissues of declining trees of Alnus glutinosa and A. incana from approximately 60 alder stands in the Czech Republic. The pathogen has been spreading rapidly in the affected alder stands, particularly in the western part of the Czech Republic. The pathogen has been frequently found in catchments of the Vltava river and Ohre river (western, northern, middle, southern Bohemia, western part of Vysocina region). Extensive decline of alder trees has not been detected in the eastern part of country (Moravia) yet, but the pathogen has been found in watercourses in the upper part of catchment of the Dyje river (Vysocina region) and is likely spreading to the east. In 2004, the causal agent of alder decline in Britain was determined to be a new hybrid, designated as Phytophthora alni subsp. alni (Brasier et al., 2004). Symptoms on diseased trees were characteristic of bleeding canker and alder decline in other regions (Jung & Blaschke, 2004): small, yellowing, and sparse foliage, dieback in the canopy and bleeding cankers on tree trunks. Many isolated cultures were similar morphologically and had characteristics that were consistent with P. alni subsp. alni (Brasier et al., 2004). Colonies growing on carrot agar (CA) were uniform, appressed with sparse aerial mycelium. Radial growth was 7–9 mm per day at 20°C on CA. Optimal growth temperature was 23–25°C , with several isolates failing to grow at 6 and 33°C. Isolates were homothallic with two-celled (22–31 × 12–20 µm) amphigynous antheridia, producing abundant terminal, spherical oogonia (28–55 µm in diam.) with moderately ornamented walls. Many oogonia aborted at a rate of 20 to 60%. Sporangiophores were simple with terminal sporangia, proliferating internally, often nested, ellipsoid in shape, measuring 38–65 × 25–41 µm (length × width ratio 1·4–1·6), and had minute papilla or were non-papillate. Comparison of DNA sequences of ITS region of two isolates (GenBank Acc. Nos EF194776, EF194777) with acquired sequences with those deposited in GenBank, confirmed their identity as P. alni. Pathogenicity was tested by artificial infection of 4-year-old black alder plants. Twenty plants were inoculated by placing segments of agar with mycelium, taken from the colony margin growing on CA, on the underbark at the collar of plants. The characteristic necroses of collars and wilting of plants were observed after several weeks and the pathogen was reisolated from damaged tissues. The control group of alder plants inoculated with sterile agar remained healthy. This is the first positive identification of P. alni with proof of its pathogenicity of black and grey alder reported in the Czech Republic.
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