Population structure and evolution of virulence in the pinewood nematode

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Five microsatellite loci were found in the nuclear genome of Bursaphelenchus xylophilus. Four of them were polymorphic and shown to help the analysis of nematode population structure. A three-year study indicated that frequencies of alleles fluctuated greatly although the mean number of alleles per locus was almost unchanged. There was a little fluctuation in the genetic diversity within a total population. The genetic diversity within nematode subpopulations sampled from individual dead trees or insect vectors decreased, which caused the genetic differentiation to increase between subpopulations. Such genetic change in nematode population had no relation to the change in the virulence (the capability of killing tree). Simultaneous inoculations of virulent and avirulent isolates on Pin us thunbergii trees showed that frequencies of alleles specific to virulent isolates were greater than those specific to avirulent isolates in diseased trees, indicating that virulent isolates proliferated more rapidly than avirulent ones. This shows that the pinewood nematode reproducing sexually satisfies one of the assumptions on which evolutionary theories of virulence by multiple infections are built in parasites and pathogens reproducing asexually. This inoculation experiment also showed appearance of new alleles possibly due to crossing over. The increase in genetic diversity may raise the difference in virulence. However, a three-year inoculation experiment by using two isolates differing in virulence showed no increase in virulence. To identify B. xylophilus and B. mucronatus, two species-specific PCR primer pairs that produce DNA fragments of different lengths were developed. Relaxation in water stress before and after the infection of B. xylophilus on P. densiflora seedlings rarely survived wilt disease.