Structure of populations of wheat powdery mildew (Erysiphe graminis DC f.sp. tritici Marchal) in Central Europe in 1993–1996: I. Dynamics of virulence

In 1993–1996, the virulence of regional populations of the wheat powdery mildew pathogen (Erysiphe graminis DC f. sp. tritici Marchal) from the Czech Republic, Austria, Hungary and Slovakia against 13 resistance genes was investigated. The populations differed mainly at the regional level. Populations from the Czech Republic, mainly from the western regions, showed higher values of virulence against the Pm4b gene. Lower frequency of virulence against Pm4b was found in Austria, and the lowest value was observed in Hungary. The differences in frequencies of virulence against Pm4a and Pm4b showed a similar geographic pattern across the four countries: a continuous decline from west to east and from north to south. Virulence against Pm2 decreased in all countries considered; virulence to pm5, Pm6, Pm8 and Mli was high throughout. Genes and gene combinations that can ensure a relatively effective biological protection against this pathogen across Central Europe at present are Pm3b, Pm2+Mld and Pm1+2+9. Czech and Slovak populations were the most complex: virulence complexity reached a maximum in Slovakia in 1994. A similar evolution, though less significant, was observed in the Czech Republic. Data on complexity of isolates suggest that Central European populations of wheat powdery mildew tend to reach an intermediate level representing the optimal number of virulence genes. This process is probably a consequence of stabilizing selection.     

泰国输华真柏中一种隐皮孢囊线虫的鉴定

深圳口岸从来自泰国真柏苗木中截获一种隐皮孢囊线虫。序列分析显示截获的孢囊线虫28S r DNA-D2/D3区基因序列与Gen Bank中布林克曼隐皮孢囊线虫(Cryphodera brinkmani)(登录号为JQ965677和DQ328705)的序列相似性为99%。系统进化树中截获的孢囊线虫和布林克曼隐皮孢囊线虫聚为一个分支。结合形态学特征比较和分子特征分析,将截获的孢囊线虫鉴定为布林克曼隐皮孢囊线虫。这是我国口岸首次报道截获该线虫。     

Molecular polymorphisms between populations of Pseudoperonospora cubensis from Greece and the Czech Republic and the phytopathological and phylogenetic implications

Molecular genetic polymorphisms within Pseudoperonospora cubensis isolates of different geographic origins were investigated to establish their phylogenetic relationships and to assess genetic variability between two distant pathogen populations. Thirty isolates originating from Greece (Crete; 15), the Czech Republic (13), the Netherlands (one) and France (one) were analysed by AFLP fingerprinting and ITS 5·8S rDNA sequence analysis. All isolates were obtained from cucumber ( Cucumis sativus ) plants showing typical downy mildew symptoms. Four AFLP primer combinations produced a total of 288 high-quality bands of which 45% were polymorphic, allowing isolates to be grouped into two separate clusters: one including the Central European (Czech Republic) and Western European (the Netherlands and France) and the other the Cretan isolates. Within each AFLP cluster there was some variation, which could be accounted for by geographic origin or pathogenicity. The two populations (Cretan vs. Central and Western European) exhibited a high degree of genetic isolation. There was no clear AFLP grouping of isolates on the basis of pathotypes. No variability was detected in the ITS1 region; however, ITS2 sequences grouped P. cubensis isolates in two subclusters: one with all investigated European and the other with Asian isolates. The two subclusters formed a larger P. cubensis cluster which was differentiated from the cluster of the neighbouring species Pseudoperonospora humuli . Within P. cubensis , AFLP fingerprints could resolve genetically isolated populations, even on small or medium geographic scales, while ITS2 sequence showed differences on a global scale, being only suitable for phylogenetic analyses.     

Population genetic structure of Mycosphaerella graminicola and Quinone Outside Inhibitor (QoI) resistance in the Czech Republic

Damage caused by the wheat pathogen Mycosphaerella graminicola increased rapidly during the last two decades in the Czech Republic. We collected isolates from naturally infected fields in seven wheat-growing locations and analysed these using eight microsatellite markers. All markers were highly polymorphic. We found a high degree of genetic diversity and low clonality within all sampled Czech populations. We identified 158 unique multilocus haplotypes among 184 isolates. Field populations showed weak genetic structure but we detected more differentiation between climatic regions within the Czech Republic. We compared the Czech field populations to populations from the United Kingdom, Germany and Switzerland and found a marked differentiation between Czech populations and Western European populations. We hypothesize that decades of different agricultural practices, including the use of different wheat cultivars, may explain this genetic differentiation. We detected a rapid increase in QoI fungicide resistance during the sampling period from 2005 to 2011, coinciding with the widespread application of this class of fungicides in the Czech Republic. M. graminicola populations in the Czech Republic underwent a rapid adaptive evolution from sensitivity to resistance similar to what was described earlier in Western Europe.     

Molecular characterization of <Emphasis Type="Italic">Xiphinema brevicollum</Emphasis> (Nematoda: Longidoridae) from the Czech Republic

Populations of Xiphinema brevicollum occurring in the Czech Republic were described morphologically and molecularly. Published species-specific primer set BL18 and BV3 was used to amplify three populations of X. brevicollum from the Czech Republic. These primers were tested against 9 species of Xiphinema and 11 species of Longidorus. Amplification was also observed for X. inaequale, X. italiae and X. lambertii. Three additional markers, mitochondrial cytochrome c oxidase subunit 1, ribosomal D2/D3 expansion segment of the 28S gene and 18S gene, were amplified and sequenced for X. brevicollum, X. inaequale and X. lambertii belonging to X. americanum-group. Comparison of cox1 sequences of X. brevicollum from the Czech Republic with X. taylori from the Slovak Republic (accession number AM086702) suggested that these populations represent the same species.     

Leek Proliferation: A New Phytoplasma Disease in the Czech Republic and Italy

During the summer 1996, twelve of twenty-eight leek plants located in a garden near eské Budjovice, South Bohemia exhibited symptoms typical of diseases associated with phytoplasmas. In summer 1998 similar symptoms were detected in leek plants in a field used for seed production located in Romagna, North Italy. In both cases the plants were established in the spring of the previous year. Plants showed flower abnormalities: stamen elongation, anther sterility, pistil proliferation, as well as poor, if any, seed production. Phytoplasma-like structures were detected by scanning and transmission electron microscopy in phloem sieve elements in the Czech diseased plants, but not in healthy ones. Nested-PCR amplifications of extracted DNA with phytoplasma-specific oligonucleotide primer pairs confirmed the presence of phytoplasmas in these plants at low concentrations. Restriction fragment length polymorphism analyses of amplified ribosomal sequences allowed the identification of detected phytoplasmas: all the samples from the Czech Republic contained aster yellows related phytoplasmas (16SrI-B) while in the Italian samples aster yellows related phytoplasmas (16SrI-B) together with stolbur related phytoplasmas (16SrXII-A) were identified. This is the first report of detection and identification of a phytoplasma disease of leek in the Czech Republic and Italy.     

Fungicide sensitivity of populations of wheat powdery mildew (Erysiphe graminis f.sp. Tritici) in Central Europe in 1993

In 1993 we observed the sensitivity of wheat powdery mildew populations (Erysiphe graminis DC f.sp. tritici Marchal) from the Czech Republic, Austria, Hungary and Slovakia to the fungicides triadimenol, tebuconazole, propiconazole, flutriafol and fenpropimorph. The highest resistance value was shown to triadimenol, which attained a mean resistance factor (MRF) of 29 (expressing how many times the population is more resistant than are standard sensitive isolates) in the mildew population from the Czech Republic. The mildew populations from eastern Slovakia and eastern Hungary, populations geographically isolated from the other populations, showed very high sensitivity to all fungicides tested. There was most sensitivity to fenpropimorph (smallest MRF values) compared with the other fungicides. Cross-resistance was established among all triazoles used, but not between triazoles and fenpropimorph. Sensitivity of wheat powdery mildew populations from Central Europe to these fungicides is considered adequate, and the development of resistance has shown a decreasing tendency in recent years.     

Characterisation of Czech arable weed communities according to management and production area considering the prevalence of herbicide-resistant species

Herbicide resistance is a widespread issue that impacts management of conventional farms, but also has ramifications for the weed community assembly; it is therefore important to see how these species factor into the weed community assembly of farms throughout the countryside. This research analysed species richness and community diversity in 98 field relevés from 48 organic and 50 conventional farms evenly distributed between two major production regions of the Czech Republic and then evaluated the incidence of species which have been reported resistant in the Czech Republic and its neighbouring countries. Farms were selected independently of any acknowledged resistant species. Out of 164 species found in this survey, only eight species have had herbicide-resistant biotypes reported in the Czech Republic, while a total of 19 species had herbicide-resistant biotypes reported in neighbouring countries. Species with recorded resistance to PSII inhibitors in the Czech Republic tended to be found together and were mostly associated with the beet production region, characterised by low altitude: Amaranthus retroflexus, Chenopodium album, Echinochloa crus-galli and Solanum nigrum. Species with reported resistance to ALS and ACCase-inhibiting herbicides were not clearly associated with a particular region or farming type. Of the species which have had reported herbicide resistance in the neighbouring countries, several were found in conventional fields within the growing season and we recommend immediate screening for herbicide resistance in these species and more diligent action in management according to anti-resistance strategies: Bromus sterilis resistant to ACCase-inhibiting herbicides, A. retroflexus resistant to ALS-inhibiting herbicides or Avena fatua resistant to ACCase and ALS-inhibiting herbicides. This work is unique in that it is evaluating weed species diversity in organic and conventional farms and using the context to frame the prevalence of high-risk herbicide-resistant species; thereby putting the potential incidence of herbicide resistance into perspective at the landscape level.     

First report of a 16SrIII-B subgroup phytoplasma associated with leaf reddening, virescence and phyllody of purple coneflower

Purple coneflower plants showing leaf reddening and flower abnormalities were observed in South Bohemia (Czech Republic). Transmission electron microscopy observations showed phytoplasmas in sieve cells of symptomatic plants but not in healthy ones. Polymerase chain reactions with universal and group specific phytoplasma primers followed by restriction fragment length polymorphism analyses of 16S rDNA allowed us to classify the detected phytoplasmas into the X-disease group, ribosomal subgroup 16SrIII-B. Sequence analyses of the 16S-23S ribosomal operon (1684 bp), ribosomal protein L15, and protein translocase genes (1566 bp) confirmed the closest relationship with phytoplasmas belonging to the 16SrIII ribosomal group, specifically the 16SrIII-B subgroup. The current study reports purple coneflower as a new host for the X-disease phytoplasma group in the Czech Republic and worldwide.     

Babesia microti (Piroplasmida: Babesiidae) in nymphal Ixodes ricinus (Acari: Ixodidae) in the Czech Republic

A total of 350 nymphs of the common tick Ixodes ricinus (Linnaeus, 1758) were collected in an endemic focus of Lyme borreliosis (South Moravia, Czech Republic) and examined for the presence of the protozoan Babesia microti (Fran?a, 1909) by polymerase chain reaction (PCR), using primers specific for the B. microti gene encoding small subunit rRNA. The assay revealed five positive pools (out of 70 pools examined); the corresponding prevalence rate was about 1.5%. Sequence analysis of the PCR products confirmed their 100% homology with that of B. microti. The study represents the first evidence of B. microti in ixodid ticks in the Czech Republic.     

Electron Microscopy and Molecular Characterization of Phytoplasmas Associated with Strawflower Yellows in the Czech Republic

Strawflower (Helichrysum bracteatum) with symptoms resembling those associated to phytoplasma infection were observed in several areas in the Czech Republic during the period 1994–2001. Plants with leaf bronzing, reddening and necrosis, proliferation of secondary shoots, flower abnormalities and dwarfing died in advanced stages of the disease. The disease incidence ranged from 2% to 70% and caused significant loss to the flower and seed production. Transmission electron microscopy showed phytoplasmas in sieve cells of affected plants, but not in healthy ones. Association of phytoplasmas with the disease was confirmed by polymerase chain reaction using phytoplasma universal ribosomal primers R16F2n/R16R2. An amplification product of the expected size (1.2 kb) was observed in all samples of the symptomatic strawflowers. The restriction profiles obtained following separate digestion with three endonucleases (AluI, HhaI, MseI) showed that phytoplasmas infecting strawflowers from different localities in the Czech Republic were uniform and undistinguishable from aster yellows (subgroup 16SrI-B). Sequence analysis of 1771 bp of the ribosomal operon amplified with primers P1/U3, R16F2n/R2 and 16R758/P7 indicated that the closest related phytoplasmas were those associated with 'Rehmannia glutinosa var. purpurea', both originating from Bohemia. This is the first report on the occurrence of a phytoplasma-associated disease of strawflower in the Czech Republic.     

Comparative molecular analysis of Bursaphelenchus vallesianus,a wood‐inhabiting nematode isolated from declining pine trees in the Czech Republic

The Bursaphelenchus genus (Nematoda: Parasitaphelenchidae) comprises mostly wood‐inhabiting nematodes that feed on various tree‐colonizing fungi. One species of the genus, B. xylophilus, has been proven as an agent causing pine wilt disease (PWD). However, involvement of other Bursaphelenchus species in the PWD remains enigmatic. In the current paper, comparative molecular analysis is performed based on nuclear ribosomal DNA (rDNA) of B. vallesianus, a species that was recently isolated from pine trees (Pinus sylvestris) exhibiting wilting and declining symptoms in the Czech Republic. Sequencing of the nuclear‐encoded ITS1–5·8S–ITS2 rDNA region confirmed previous taxonomic conclusions based on morphology. Evolutionary reconstructions resulted in a phylogenetic tree, where the Czech isolate of B. vallesianus occupied a common clade together with other species belonging to the so‐called B. sexdentati group. Unexpectedly, comprehensive analysis of the sequence data revealed a genetic variation distinguishing the Czech isolate of B. vallesianus from all other species of the B. sexdentati group. This dissimilarity consists of the presence of a four nucleotide exchange found in the 5·8S rRNA‐coding gene. The newly identified genetic variation appears to affect the 5·8S rRNA folding, as deduced from secondary structure models. Additionally, it is shown that for the first time, to the authors’ knowledge, both bursaphelenchid internal transcribed spacers (ITS1 and ITS2) fold into the multibranched closed loops. While the ITS2 closed loop is formed with help of canonical 5·8S‐28S rRNA pairing, the ITS1 forms the thermodynamically stable closed loop with no support of flanking rRNA sequences. The current information on bursaphelenchid ITS rDNA sequence diversity and structure is further discussed.     

Surveys for Phytophthora ramorum in the Czech Republic1

The State Phytosanitary Administration (SPA) of the Czech Republic has been conducting an official survey for Phytophthora ramorum since 2003. A single outbreak of P. ramorum was detected by the SPA on guelder rose (Viburnum bodnantense) in autumn 2003 during a phytosanitary inspection in an ornamental nursery made on the request of owner of the nursery as he had noticed plants of Viburnum bodnantense withering. The infested lot consisted of 15 plants, which were imported from Italy into the Czech Republic at the end of April in 2003. A post‐entry inspection was carried out at the place of destination just after arrival and no visual symptoms were seen at that time. The lot was destroyed by the owner of the nursery immediately after the sampling. A survey was carried out by the SPA in all nurseries in which host plants coming from the same origin as the infested plants had been planted. No other outbreak was found. Phytosanitary inspections were carried out in ornamental nurseries and garden centres, public parks and forestry sites. No outbreak was detected in 2004 (451 visual inspections) and 2005 (522 visual inspections). The status of Phytophthora ramorum in the Czech Republic is: absent, one isolated outbreak linked to an imported consignment was eradicated.     

Hymenopterous parasitoids of the cherry bark tortrix, Enarmonia formosana (Scopoli) in central-east Europe (Hymenoptera, Ichneumonoidea; Lepidoptera, Tortricidae)

Strategies targeting the biological control of the cherry bark tortrix (=CBT), Enarmonia formosana (Scopoli) in North America have also included the search for potentially useful native hymenopterous parasitoids of the pest in Europe. The search for such agents undertaken in the central-east Europe (Czech Republic, Slovak Republic, Hungary) resulted in determination of several ichneumonid species (Campoplex cf. dubitator Horst., Liotryphon sp., Lissonotus sp., Pimpla spuria (Grav.), Pimpla turionellae (L.), and some others, and a braconid (Apanteles britannicus Wilk.) in 2001–2002.     

Hymenopterous parasitoids of the cherry bark tortrix, Enarmonia formosana (Scopoli) in central-east Europe (Hymenoptera, Ichneumonoidea; Lepidoptera, Tortricidae)

Strategies targeting the biological control of the cherry bark tortrix (=CBT), Enarmonia formosana (Scopoli) in North America have also included the search for potentially useful native hymenopterous parasitoids of the pest in Europe. The search for such agents undertaken in the central-east Europe (Czech Republic, Slovak Republic, Hungary) resulted in determination of several ichneumonid species (Campoplex cf. dubitator Horst., Liotryphon sp., Lissonotus sp., Pimpla spuria (Grav.), Pimpla turionellae (L.), and some others, and a braconid (Apanteles britannicus Wilk.) in 2001–2002.     

Confirmation of <Emphasis Type="Italic">Peronospora agrimoniae</Emphasis> as a distinct species

Leaves with typical symptoms of downy mildew were found on common agrimony in the Czech Republic in 2014 and 2015 and at several locations in Germany from 2010 to 2014. The causal agent of downy mildew of agrimony was often reported as Peronospora agrimoniae, but sometimes also as P. sparsa. Morphological characteristics of the pathogens found in both countries are in the range of previous works for P. agrimoniae, but also other downy mildews parasitic on Rosaceae, rendering their discrimination based on published observations difficult. For molecular identification sequencing of several loci (nrITS rDNA, cox1 and cox2) was performed. Phylogenetic analyses based on nrITS rDNA clearly separated P. agrimoniae from other Peronospora species infecting Rosaceae. Thus, considering P. agrimoniae as separate species seems justified. Two German specimens were identical to two Czech samples in both nrITS rDNA and cox1 mtDNA sequences, but differed in a single nucleotide substitution in cox2 region. To our knowledge, this is the first verified record of P. agrimoniae on common agrimony in the Czech Republic.     

Distribution of the double-spined spruce bark beetle <Emphasis Type="BoldItalic">Ips duplicatus</Emphasis> in the Czech Republic: spreading in 1997–2009

Ips duplicatus (Coleoptera: Curculionidae) was reported in the Euro-Siberian taiga from Sweden to Sakhalin and in the Alps at the beginning of the 20th Century. It has been recorded in spruce stands in the north-eastern part of the Czech Republic since the beginning of the 1970s; monitoring of I. duplicatus was conducted in the Czech Republic in 9 years between 1997 and 2009. Beetles of the overwintering generation were captured in Theysohn pheromone traps baited with a lure of ipsdienol and E-myrcenol, and after 2005 in ID Ecolure lures. Surprisingly, I. duplicatus was found throughout the Czech Republic already in 1997. In 2002, there was a nationwide decrease in their numbers. Particularly since 2005, there has been a spread from the main center of occurrence in the eastern areas to the south and west into the central part of Bohemia where, until that time, its population densities had been lower and had not caused economically significant damage. Moreover, it was observed that in the north-eastern parts of the Czech Republic up to 80% of spruce trees from sanitary felling had been infested by this pest in recent years. The recorded amount of spruce trees infested by I. duplicatus is correlated significantly with the amount of spruce trees recorded as damaged by Ips typographus, Ips amitinus and Pityogenes chalcographus infestation as well as with the numbers of individuals of I. duplicatus captured during the aforementioned monitoring.