Introduction

Abstract

Following the discovery that the pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (Kiyohara and Tokushige 1971), researchers focused on identifying the wilting mechanism. In recent years that effort has been aided by dramatic improvements in the various techniques and other technological advancements that have been employed in these studies. The purpose of the authors here is to discuss some of those techniques and advancements as related to wilting of host pines. The artificial propagation of PWN and an inoculation technique were first established by Mamiya (1980, 1984b), and then physiological measurements were conducted (Tamura et al. 1987, 1988). During the 1980s, highly virulent PWNs were selected from many cultures obtained from dead pines (Kiyohara and Dozono 1986; Kiyohara and Bolla 1990). It is very important to conduct inoculation tests with virulent races of PWN that successfully kill pine trees, as observed during natural infection. Early studies used trees 10-20 cm in diameter later while smaller and younger saplings less than 5 years old were used (Mamiya 1985). Supported by these fundamental achievements, detailed investigations to clarify the wilting mechanism have been conducted in Japan since the 1980s (Tamura and Dropkin 1984; Tamura et al. 1988). By the end of the 1990s hundreds of reports had been published on the phenomena related to trees inoculated with the PWN (Fukuda 1997; Yamada 2006) as well as reports on incidents associated with natural infection and the behavior of vector beetles (Kishi 1995).