The quarantine nematode Bursaphelenchus xylophilus (pine wood nematode, PWN) is the causal agent of pine wilt disease in Asia and Europe. Great variation has been reported in the virulence level among populations of B. xylophilus. It is difficult to distinguish nematodes differing in virulence based on their morphology, therefore molecular biological techniques have been developed to identify virulence of various B. xylophilus populations. The real-time PCR-HRM was developed and evaluated as a method to detect and distinguish virulent and avirulent populations of B. xylophilus. A set of primers was designed to target the ITS-2 region of rDNA. Reliability of the newly developed primer pair was examined and positively verified on eight virulent and two avirulent isolates of B. xylophilus originating from geographically distant localities in Europe and Asia. The results showed two distinct melting curve profiles related to each of the examined virulent forms. The results have demonstrated that the real-time PCR-HRM analysis is a rapid, simple and accurate method to detect and distinguish virulent and avirulent populations of B. xylophilus.

Bursaphelenchus xylophilus jest czynnikiem sprawczym choroby więdnięcia sosny, doprowadzającym do zamierania drzew sosnowych na kontynencie azjatyckim, a od 1999 roku również w Europie. Dotychczasowe badania prowadzone nad patogenicznością nicienia B. xylophilus wykazały, że nie wszystkie izolaty tego szkodnika doprowadzają do zamierania drzew. W obrębie jego populacji stwierdzono występowanie patogenicznych, jak i całkowicie niepatogenicznych jego izolatów. Morfologiczne odróżnienie poszczególnych populacji charakteryzujących się różną patogenicznością jest całkowicie niemożliwe, dlatego poszukiwane są metody molekularne umożliwiające jak najbardziej precyzyjne ich odróżnienie. W przeprowadzonych badaniach do odróżniania pato-genicznych i niepatogenicznych populacji tego kwarantannowego nicienia wykorzystano reakcję real-time PCR-HRM. W badaniach wykorzystano całkowite DNA patogenicznych oraz niepatogenicznych populacji B. xylophilus pochodzących z różnych rejonów świata. Dla populacji tych zaprojektowano własne startery, które amplifikowały fragment regionu ITS-2 rDNA. Przeprowadzone doświadczenia potwierdziły wysoką skuteczność zaprojektowanych starterów do odróżniania patogenicznych i niepatogenicznych populacji kwarantannowego nicienia B. xylophilus.

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