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Optimization of mycovirus detection methods obtained from NGS data analysis
PÍCHALOVÁ, Barbora
One well-known pathogenic fungus Armillaria causing Armillaria root disease which is characterised by infecting the root system of woody plants, resulting in basal decay and the development of a white syrrocium under the bark of trees. Several genome sequences of mycoviruses have already been published in research on this disease, including viruses from the Mymonoviridae, Botourmiaviridae, Partitiviridae, Virgaviridae as well as a newly described group of viruses known as ambi-like viruses. In this study, alongside ambi-like viruses and partitiviruses, by in silico analysis of virus sequences obtained via NGS methods unveiles the identification of previously unknown beny-like viruses within Armillaria spp. from the Czech Republic. To confirm the presence of beny-like viruses in vitro, reverse transcription with PCR was used. Total RNA was isolated using a commercial kit and its presence in the samples was confirmed electrophoretically. The sequence obtained using the Illumina system's NovaSeq 6000 sequencing platform was used to design the triple pairs of primers Beny F1 and Beny R1, Beny F2 and Beny R2, and BV1 F and BV1 R, in which potential ORFs were designed. The primers were designed to target the longest ORF encoding RdRp, the most conserved gene in RNA viruses. The DNA fragment obtained by reverse transcription was amplified by PCR followed by reamplification. Finally the samples were evaluated electrophoretically for the presence of viral single-stranded RNA.
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Molecular identification of Armillaria
SALZMANOVÁ, Kateřina
Armillaria species causes economic losses in forestry in the Czech Republic. Combination of drought stress, Armillaria infection and bark beetles (Ips typographus) is the main reason on tree mortality in Norway spruce (Picea abies) plantation. The theoretical part describes genus Armillaria. There is desribed the role in Norway spruce mortality and the controle of Armillaria root rot of trees. Review also includes molecular markers used in Armillaria identification and description and principle of methods used in this study. The aim of this thesis was identification of Armillaria species came from drought stressed forrests from the Czech Republic. Molecular analysis was based on polymorphisms in the translational elongation factor alpha gene (EF 1 alpha). The empirical part presents the results of the study. The studied population consisted 92 samples of Armillaria DNA came from drought stressed forrests. The results confirmed the highest incidence of Armillaria ostoyae (66 samples). There were identified also Armillaria cepistipes (22 samples) and Armillaria gallica (5 samples). The results was used to build phylogenetic tree.
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Distribution of Heterobasidion and Armillaria root rots in Vallombrosa fir forest, Italy
Dálya, László Benedek
This work intends to describe the present condition of Vallombrosa forest (Tuscany, Italy) from the phytopathological point of view. The chronic disease caused by Heterobasidion and Armillaria root rots is a key factor affecting the vitality of silver fir plantations of the region. Detailed knowledge about their distribution could help to control the pathogens. Systematic sampling and survey of damages on trees were undertaken at 52 points. Identification of different species from soil and fungal samples was accomplished by DNA-based methods (TSCP, nested PCR, RFLPs analysis). The high presence of both parasitic fungi was detected under a wide range of ecological conditions. Data analysis indicates the strong spreading potential of the pathogens even into new habitats, especially in connection with water stress of their hosts.
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Investigations of fungal pathogens of Douglas-fir on various provenance plots
Lísková, Aneta ; Pešková, Vítězslava (advisor) ; Vachová, Jana (referee)
The bachelor thesis evaluates the incidence of fungal pathogens due to various provenance of Pseudotsuga menziesii Mirbel, Franco. The research was conducted on the provenance area Jizbice - LS Vlašim. In this area, the presence of fungus of the genus Armillaria was evaluated. By using standard phytopathological methods, samples of needles and offshoots were taken, and the occurrence of the various needle blight species was investigated. In the field the preliminary determination and quantification of the occurrence of fungus pathogens infestation were implemented. Consequently, the microscope method was used for the fungal species spectrum completion and specification in the laboratory. The fungus Rhabdocline pseudotsugae was discovered. The fungus Rhizosphaera genus was discovered on some needles from the researched provenances. The occurrence of the Armillaria spp. was not confirmed.
It was determined, that it highly depends on the provenance of the infested tree. The most resistant provenance was Nimkish (1025), located in the north part of the Vancouver island, British Columbia. The least resistant provenance, in terms of defoliation, was provenance Merritt (1028) from British Columbia. Correspondence analysis results showed that the provenance of 1010 and 1028 showed the highest number of individuals and can be expected to have better resistance to abiotic and biotic effects of harmful factors.
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