Demonstration of interbreeding between virulent and avirulent populations of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) by PCR-RFLP method

Abstract

The pinewood nematode, Bursaphelenchus xylophilus, is the causative agent of pine wilt disease. The virulence of nematodes significantly varies among pine trees, but does not vary within a single pine. To elucidate the reason for no variation in virulence within a single pine, a technique to investigate the population structure of nematodes of different virulence is needed. In this study, the demonstration of interbreeding between virulent and avirulent populations of B. xylophilus in vitro was attempted using PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. Ribosomal DNA containing the 5.8S gene, the internal transcribed spacer regions 1 and 2, and partial regions of 18S and 28S genes were used for analysis. First, PCR-RFLP patterns of offspring produced by interbreeding between two individuals of virulent and avirulent isolates were analyzed. The offspring from a single-pair interbreeding showed 3-digested fragment patterns with the restriction enzyme HhaI, patterns the same as the virulent isolate, the avirulent isolate and a hybrid pattern containing both fragments of the virulent and avirulent isolates. The virulent population was mixed with the avirulent one in vitro and propagated nematodes were individually analyzed by PCR-RFLP method. The nematodes showed the same 3 PCR-RFLP patterns as the offspring from a single-pair interbreeding. The detection of nematodes with a hybrid pattern demonstrates the occurrence of interbreeding between virulent and avirulent populations of B. xylophilus.