Diversity and ecology of Armillaria species in virgin forests in the Ukrainian Carpathians

In this study, we investigated the diversity and ecology of Armillaria species in virgin pure beech and mixed conifer forests (15,000ha) of the Carpathian Biosphere Reserve in Ukraine. Armillaria rhizomorphs were systematically sampled, both from the soil and from the root collar of trees (epiphytic), on 79 plots (25 × 20m) of a 1.5 × 1.5km grid. In both forest massifs, rhizomorphs were present in the majority of the soil samples, with an estimated dry weight of 512kg/ha in the pure beech forests and 223kg/ha in the mixed conifer forests. Similarly, in both forest massifs, most of the trees inspected had rhizomorphs at the root collar. Species identification based on DNA analyses showed that all five annulated European Armillaria species occur in these virgin forests, as previously observed in managed forests in central Europe. However, differences in the frequencies of the single species were observed. The predominance of the preferentially saprotrophic A. cepistipes and A. gallica (84 and 15% of the specimens, respectively) and the absence of significant pathogenic activity suggest that in these virgin forests Armillaria species are most likely to behave as saprotrophs. Forest management may increase the frequency of the pathogenic species A. ostoyae, which is rare in virgin forest

Eight microsatellite markers for Armillaria cepistipes and their transferability to other Armillaria species

We isolated eight polymorphic microsatellite markers for the basidiomycete Armillaria cepistipes and characterised them by analysing 50 isolates representing two geographically distinct populations from Switzerland and the Ukraine. The number of alleles per locus and population varied from one to eight, resulting in 43 alleles over the eight loci and two populations. In both populations, no significant linkage disequilibrium was observed between pairs of loci. Significant (P < 0.05) deviations from Hardy-Weinberg equilibrium were observed at one locus in the Swiss population and at three loci in the Ukrainian population. Of the eight loci developed for A. cepistipes, six were also polymorphic in A. gallica, four in A. ostoyae, two in A. mellea, and one in A. borealis. Beside the potential to be used for population genetic studies on A. cepistipes, these microsatellites thus represent additional molecular markers for three of the four annulated Armillaria species occurring in Europ

Genetic diversity of the Chestnut blight fungus Cryphonectria parasitica and its associated hypovirus in Portugal

The European chestnut (Castanea sativa Mill.) agro‐ecosystem has been of high social, economic, and landscape importance in Portugal. Chestnut blight caused by the fungus Cryphonectria parasitica is considered a major cause of the decline of chestnut trees across Europe. C. parasitica is an ascomycete (Diaporthales) that is native to eastern Asia. Infection of chestnut trees with this pathogen is typically associated with extensive bark necrosis (so‐called cankers) on stems and branches, resulting in the subsequent death of the part of the tree above the infection point. Chestnut blight in Portugal was first reported in 1992 and since expanding in distribution. Here, we investigated the invasion history of C. parasitica and its associated hypovirus in Portugal. For this, we characterized 137 isolates collected between 2013 and 2014 in four chestnut stands for virus‐infection, vegetative compatibility (vc) type, mating type and microsatellite haplotype. A total of 33 haplotypes and four vc types were observed, although the Portuguese C. parasitica population is currently dominated by a single haplotype and a single vc type (EU‐11). Further diversification may be expected due to ongoing sexual recombination, but eventually also to new migration and additional introductions. Cryphonectria hypovirus 1 (CHV‐1) was found in two populations. Genetic analysis of the six CHV‐1 isolates obtained revealed that three viral strains belong to the Italian subtype and three to the French subtype, which suggest different, independent introductions.info:eu-repo/semantics/publishedVersio

Biological control of chestnut blight. Detection, identification and characterization of the Hypovirus - CHV1

O cancro do castanheiro é provocado pelo fungo Cryphonectria parasitica é considerado a principal causa do declínio dos castanheiros na Europa. C. parasitica é um ascomiceta (Diaporthales) nativo do continente asiático. A infeção do castanheiro manifesta-se pelo aparecimento de necroses extensas na casca dos ramos e troncos, que resulta na morte dos castanheiros. A hipovirulência é um método seletivo de controlo biológico do Cancro do Castanheiro, trata-se de uma infeção do fungo C. parasitica com Cryphonectria hypovirus 1 (CHV1), um vírus de dsRNA não encapsulado do género Hypovirus. O controlo biológico com estirpes hipovirulentas é considerado um método eficaz no controlo da doença e promove a recuperação dos castanheiros. A aplicação necessita da produção em laboratório de estirpes hipovirulentas compatíveis com a estirpe virulenta presente no campo e a sua introdução na margem de cancros ativos. Neste estudo pretendeu-se detetar, identificar e caracterizar o hipovirus CHV1 com potencial para integrar programas de controlo biológico do Cancro do castanheiro em Portugal. Com a utilização de métodos moleculares foram identificados e caracterizados 11 hipovirus em isolados brancos de C. parasitica obtidos em cancros no nordeste de Portugal.The chestnut blight caused by the fungus Cryphonectria parasitica is considered a major cause of the decline and disappearance of chestnut trees across Europe. C. parasitica is an ascomycete fungus (Diaporthales) that is native to eastern Asia. Infection of chestnut plants with this fungus is typically associated with extensive necrosis (cankers) of the bark on stems and branches, resulting in the subsequent death of the tree. Hypovirulence is a specific method for biological control of Chestnut Blight, it is an infection of C. parasitica with Cryphonectria hypovirus 1 (CHV1), a unencapsidate dsRNA virus of the genus Hypovirus. Biological control with hypovirulent strains is considered an efficient method to control the disease and improve the host plant recovery. The field application needs laboratorial production of compatible hypovirulent strain and introduction by inoculation at the margin of active cankers. In this work it was intended to identify and characterize the hypovirus with potential ability to integrate biological control programmes against Chestnut Blight. Using molecular techniques 11 hypovirus with hypovirulence potential were identified on white isolates of C. parasitica isolated on cankers from northeast of Portugal

Characterization of hypovirulent isolates of the chestnut blight fungus, Cryphonectria parasitica from the Marmara and Black Sea regions of Turkey

Chestnut blight caused by the introduced fungus Cryphonectria parasitica has been responsible for the decline of Castanea sativa in Turkey since the 1960s. In this study, 72 C. parasitica isolates were recovered from the Marmara and Black Sea regions of Turkey showing white or cream-coloured culture morphology and were subjected to various tests to determine if they were infected by Cryphonectria hypovirus 1 (CHV-1). The vast majority of the isolates (69 out of 72) were vc type EU-1. Both mating types were found among a subsample of the isolates. The hypovirus was detected in 55 isolates by dsRNA extraction and/or virus specific RT-PCR on total RNA extracts. All but one isolates showed no or only weak phenol oxidase activity on agar medium containing tannic acid, typical of CHV-1 infected isolates. Through sequencing of a specific region of the hypovirus genome, we found that 24 hypovirus isolates belonged to the CHV-1 subtype I and six to the CHV-1 subtype F2. The distribution of the two CHV-1 subtypes in Turkey showed a clear geographic pattern. CHV-1 subtype I was only detected in the Marmara and western Black Sea region, whereas subtype F2 was restricted to the eastern part of the Black Sea region. The effectiveness of 23 hypovirulent isolates was tested against a virulent isolate on 2-3years old chestnut sprouts. Ten hypovirulent isolates, all infected by CHV-1 subtype I, prevented canker development by more than 80% suggesting that they might be suitable for biological control of chestnut blight in Turke

Eficácia do tratamento do Cancro do Castanheiro em ensaios de inoculação com estirpes hipovirulentas de Cryphonectriaparasitica(CHV1) em Trás-os-Montes (Portugal)

O castanheiro europeu (Castanea sativa Mill) é a principal espécie do sistema agroflorestal nas regiões montanhosas de Portugal. O castanheiro é atualmente afetado por vários agentes patogénicos agressivos, destacando-se o fungo Cryphonectria parasitica, que causa o Cancro do Castanheiro, que provoca a morte da árvore. A Hipovirulência, associada à presença de Cryphonectria hypovirus 1 no fungo fitoparasita, é um método específico e eficiente no controlo biológico do Cancro do Castanheiro. Para uma aplicação eficaz deste método no campo é necessário conhecer as características das estirpes agressivas de C. parasitica presentes no campo. Na Serra da Padrela foi selecionado um souto para realizar este estudo. Foi efetuado uma caracterização dos cancros ativos, e posteriormente o seu tratamento por Hipovirulência. Dos isolados obtidos dos cancros, 99% eram agressivos, sendo identificados 3 isolados como hipovirulentos. Todos foram classificados como vc-type EU-11, estando em maior abundância o mating-type 2, numa proporção de 2:1 comparativamente ao mating-type 1. Os cancros agressivos foram tratados com as estirpes hipovirulentas compatíveis tendo-se observado ao fim de 1 ano uma recuperação das árvores afetadas tendo-se confirmado a presença das estirpes hipovirulentas em cancro anteriormente agressivos

Caracterização da estrutura populacional de Cryphonectria parasitica para aplicação da luta biológica por hipovirulência: um caso de estudo na Serra da Padrela

O castanheiro europeu (Castanea sativa Mill) é a principal espécie do sistema agroflorestal nas regiões montanhosas de Portugal. O castanheiro é atualmente afetado por vários agentes patogénicos agressivos, destacando-se o fungo Cryphonectria parasitica, que causa o Cancro do Castanheiro, que provoca a morte da árvore. A Hipovirulência, associada à presença de Cryphonectria hypovirus 1 no fungo fitoparasita, é um método específico e eficiente no controlo biológico do Cancro do Castanheiro. Para uma aplicação eficaz deste método no campo é necessário conhecer as características das estirpes agressivas de C. parasitica presentes no campo. Na Serra da Padrela foi selecionado um souto para realizar este estudo. Foi efetuado uma caracterização dos cancros ativos, e posteriormente o seu tratamento por Hipovirulência. Dos isolados obtidos dos cancros, 99% eram agressivos, sendo identificados 3 isolados como hipovirulentos. Todos foram classificados como vc-type EU-11, estando em maior abundância o mating-type 2, numa proporção de 2:1 comparativamente ao mating-type 1. Os cancros agressivos foram tratados com as estirpes hipovirulentas compatíveis tendo-se observado ao fim de 1 ano uma recuperação das árvores afetadas tendo-se confirmado a presença das estirpes hipovirulentas em cancro anteriormente agressivos

Population History and Pathways of Spread of the Plant Pathogen Phytophthora plurivora

Human activity has been shown to considerably affect the spread of dangerous pests and pathogens worldwide. Therefore, strict regulations of international trade exist for particularly harmful pathogenic organisms. Phytophthora plurivora, which is not subject to regulations, is a plant pathogen frequently found on a broad range of host species, both in natural and artificial environments. It is supposed to be native to Europe while resident populations are also present in the US. We characterized a hierarchical sample of isolates from Europe and the US and conducted coalescent-, migration, and population genetic analysis of sequence and microsatellite data, to determine the pathways of spread and the demographic history of this pathogen. We found P. plurivora populations to be moderately diverse but not geographically structured. High levels of gene flow were observed within Europe and unidirectional from Europe to the US. Coalescent analyses revealed a signal of a recent expansion of the global P. plurivora population. Our study shows that P. plurivora has most likely been spread around the world by nursery trade of diseased plant material. In particular, P. plurivora was introduced into the US from Europe. International trade has allowed the pathogen to colonize new environments and/or hosts, resulting in population growth

Conformational Properties of a Peptidic Catalyst: Insights from NMR Spectroscopic Studies

Peptides have become valuable as catalysts for a variety of different reactions, but little is known about the conformational properties of peptidic catalysts. We investigated the conformation of the peptide H-dPro-Pro-Glu-NH; 2; , a highly reactive and stereoselective catalyst for conjugate addition reactions, and the corresponding enamine intermediate in solution by NMR spectroscopy and computational methods. The combination of nuclear Overhauser effects (NOEs), residual dipolar couplings (RDCs), J-couplings, and temperature coefficients revealed that the tripeptide adopts a single predominant conformation in its ground state. The structure is a type I β-turn, which gains stabilization from three hydrogen bonds that are cooperatively formed between all functional groups (secondary amine, carboxylic acid, amides) within the tripeptide. In contrast, the conformation of the enamine intermediate is significantly more flexible. The conformational ensemble of the enamine is still dominated by the β-turn, but the backbone and the side chain of the glutamic acid residue are more dynamic. The key to the switch between rigidity and flexibility of the peptidic catalyst is the CO; 2; H group in the side chain of the glutamic acid residue, which acts as a lid that can open and close. As a result, the peptidic catalyst is able to adapt to the structural requirements of the intermediates and transition states of the catalytic cycle. These insights might explain the robustness and high reactivity of the peptidic catalyst, which exceeds that of other secondary amine-based organocatalysts. The data suggest that a balance between rigidity and flexibility, which is reminiscent of the dynamic nature of enzymes, is beneficial for peptidic catalysts and other synthetic catalysts