Two virulent isolates and two avirulent isolates of Bursaphelenchus xylophilus and one isolate of B. mucronatus were used to investigate the relationships between life history parameters, rate of population increase and virulence. The results showed that on fungal cultures of Botrytis cinerea, virulent B. xylophilus completed one generation much faster than did avirulent B. xylophilus and B. mucronatus. There was a tendency that virulent B. xylophilus isolates laid more eggs during the egg laying period than did avirulent populations. Shorter generation time and higher fecundity resulted in a higher rate of population increase. Generation time and fecundity were primary factors determining rate of population increase. Difference in rate of population increase is closely related to variation of virulence: virulent B. xylophilus increased population size at the fastest rate, avirulent B. xylophilus was slower, and B. mucronatus was slowest. It is assumed that similar variations in life history parameters and rate of population increase are also expressed in pine trees and help to explain variation of virulence in the field.

The occurrence of cavitation events and embolism during the latent, early stage and the late developmental stages of pine wilt disease was monitored nondestructively by acoustic emission (AE) and high-resolution magnetic resonance microscopy, respectively, and the results were compared with changes in leaf water potential and stem thickness. In the latent stage of the disease, when no embolisms were observed, cavitation events were detected by AE during the daytime in water-stressed Japanese black pine (Pinus thunbergii Parl.) seedlings, indicating that cavitation occurred at the individual tracheid level. In the early stage of the disease, an increase in the frequency of AE events occurred coincidentally with the occurrence of patchy embolisms at the mass tracheid level. The threshold water potential for such mass cavitation was higher than that causing cavitation of individual tracheids during the latent stage of the disease. In the advanced stage of the disease, explosive AE events were observed coincidentally with drastic enlargement of embolized areas and decreases in water potential. The AE events in the latent stage occurred only during the daytime, whereas, in the early and advanced stages of the disease, they also occurred at night. The explosive occurrence of cavitation in the advanced stage was thought to be a case of "runaway embolism."

The development of xylem cavitation caused by pine wilt disease was visualized nondestructively with a compact magnetic resonance (MR) microscope system. A T(1)-weighted spin-echo sequence clearly visualized the water-filled xylem of Japanese black pine (Pinus thunbergii) as white zones, whereas cavitated xylem was represented as dark areas. Cavitated areas in the xylem were first observed 6 to 9 days after inoculation with the pinewood nematode (Bursaphelenchus xylophilus), and enlarged gradually over several days. After 11 to 18 days, cavitated areas rapidly increased in size, fused, and reached the cambium. This drastic expansion in cavitation coincided with and appeared to explain the sudden wilting of the seedlings. The development of cavitation observed through MR microscopy corresponded well with previous descriptions of disease progression.

We analyzed the genetic structure of Bursaphelenchus xylophilus populations within individual trees (subpopulations) in three distant pine forests (Tanashi, Tsukuba, and Chiba in Japan) based on the polymorphism of four microsatellite (SSR) markers. Most of the nematodes from subpopulations in Tanashi showed the same genotype over 2 years, indicating that nematodes of that genotype dominated there for years. In contrast, 16 and 15 genotypes were identified in nematode populations from Tsukuba and Chiba, respectively. Despite the high genetic diversity within the Tsukuba and Chiba populations, extremely low genetic diversity was observed within the subpopulations. The genetic difference between the Tsukuba and Chiba populations was significantly smaller than that between Tanashi and either Tsukuba or Chiba. Observed heterozygosity was significantly less than expected based on Hardy-Weinberg equilibrium. These findings are best explained by a founder effect, geographic isolation between populations, explosive nematode multiplication from a small number within individual trees, and the Wahlund effect.

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