Molekularna diagnostyka wybranych patogenów z rodzaju Phytophthora w ramach integrowanej ochrony roślin

• Autorzy: Nowakowska J.A. , Malewski T. , Tereba A. , Borys M. , Oszako T.

Warianty tytułu

EN

Molecular diagnostic of Phytophthora pathogens as a tool for Integrated Pest Management

• Języki publikacji: PL

Abstrakty

EN

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.

Słowa kluczowe

PL

fitopatologia; Phytophthora; wykrywanie; identyfikacja; Phytophthora alni subsp.multiformis; Phytophthora lacustris; Phytophthora taxon hungarica; metody badan; metoda real time PCR; sondy TaqMan

• Wydawca: -
• Czasopismo: Sylwan
• Rocznik: 2016
• Tom: 160
• Numer: 05
• Opis fizyczny: s.365-370,rys.,tab.,wykr.,bibliogr.

Twórcy

autor

Nowakowska J.A.

• Laboratorium Biologii Molekularnej, Instytut Badawczy Leśnictwa, Sękocin Stary, ul.Braci Leśnej 3, 05-090 Raszyn

autor

Malewski T.

• Muzeum i Instytut Zoologii, Polska Akademia Nauk, ul.Wilcza 64, 00-679 Warszawa

autor

Tereba A.

• Laboratorium Biologii Molekularnej, Instytut Badawczy Leśnictwa, Sękocin Stary, ul.Braci Leśnej 3, 05-090 Raszyn

autor

Borys M.

• Laboratorium Biologii Molekularnej, Instytut Badawczy Leśnictwa, Sękocin Stary, ul.Braci Leśnej 3, 05-090 Raszyn

autor

Oszako T.

• Zamiejscowy Wydział Leśny w Hajnówce, Politechnika Białostocka, ul.Piłsudskiego 8, 17-200 Hajnówka

Bibliografia

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