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    Tytuł:
    Multipleksowa detekcja gatunków Phytophthora przy użyciu platformy Fluidigm
    Multiplex detection of Phytophthora spp. using the Fluidigm platform
    Autorzy:
    Sikora, K.
    Oszako, T.
    Kubiak, K.
    Nowakowska, J.A.
    Malewski, T.
    Powiązania:
    https://bibliotekanauki.pl/articles/2142612.pdf
    Data publikacji:
    2020
    Wydawca:
    Instytut Badawczy Leśnictwa
    Tematy:
    forest soil
    oomycetes
    oak dieback
    pathogens
    next generation sequencing
    gleby leśne
    oomycety
    zamieranie dębów
    patogeny
    sekwencjonowanie nowej generacji
    Opis:
    The genus Phytophthora plays an important role not only in agriculture but also in forest ecosystems. As the number of known Phytophthora species continues to grow, identifying new isolates in this genus has become increasingly challenging even by DNA sequencing. Therefore, the development of proper techniques for detection and identification is crucial for monitoring and control of these pathogens in the forestry sector. In recent years, new molecular methods using innovative approaches have indeed been developed. However, the majority of these methods was designed to detect single Phytophthora species. Techniques that are able to target multiple species would offer advantages, especially for the assessment of Phytophthora diversity in the environment. This paper describes a multiplex assay for the identification of eight Phytophthora isolates, down to the species level, based on a Fluidigm platform employing pyrosequencing. The obtained results showed that for an accurate determination of the species, it is sufficient to know the sequence of two markers, ITS and COX1. The sensitivity of this test is sufficient to identify Phytophthora in a pure culture. Unfortunately, analysis based on a pyrosequencing platform does not provide enough data to simultaneous identify multiple Phytophthora species in samples collected in the field. This problem could be resolved in the future by sequencing using more efficient platforms like Illumina or IonTorrent.
    Źródło:
    Leśne Prace Badawcze; 2020, 81, 4; 161-166
    1732-9442
    2082-8926
    Pojawia się w:
    Leśne Prace Badawcze
    Dostawca treści:
    Biblioteka Nauki
    Artykuł
    Tytuł:
    Molekularna diagnostyka wybranych patogenów z rodzaju Phytophthora w ramach integrowanej ochrony roślin
    Molecular diagnostic of Phytophthora pathogens as a tool for Integrated Pest Management
    Autorzy:
    Nowakowska, J.A.
    Malewski, T.
    Tereba, A.
    Borys, M.
    Oszako, T.
    Powiązania:
    https://bibliotekanauki.pl/articles/989551.pdf
    Data publikacji:
    2016
    Wydawca:
    Polskie Towarzystwo Leśne
    Tematy:
    fitopatologia
    Phytophthora
    wykrywanie
    identyfikacja
    Phytophthora alni subsp.multiformis
    Phytophthora lacustris
    Phytophthora taxon hungarica
    metody badan
    metoda real time PCR
    sondy TaqMan
    real time pcr
    taqman
    butt and fine root pathogens
    phytophthora
    Opis:
    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 R² 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 phytophthoras 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.
    Źródło:
    Sylwan; 2016, 160, 05; 365-370
    0039-7660
    Pojawia się w:
    Sylwan
    Dostawca treści:
    Biblioteka Nauki
    Artykuł
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