Two Cyclic Dipeptides from Pseudomonas fluorescens GcM5-1A Carried by the Pine Wood Nematode and Their Toxicities to Japanese Black Pine Suspension Cells and Seedlings in vitro

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD). It is native to North America and has been introduced to Japan by way of timber trade in the early 20th century, from where it has spread into China and Korea. In 1999, it was reported for the first time in Portugal and in Europe (Mota et al., 1999) and has become a worldwide threat to forest resources. It is essential to know the mechanism of pine wilt caused by PWN, in order to better control the nematode. Bursaphelenchus xylophilus has been thought to be the only pathogen of PWD (Mamiya, 1983; Myers, 1988; Yang, 2002), though the mechanism of the disease has not been well elucidated. However, there has been an increase in claims that bacteria are associated with PWN, and thus surface-sterilized PWN would lose its pathogenicity (Kawazu and Kaneko, 1997; Oku et al., 1980; Zhao et al., 2000a). Each nematode from naturally infected Japanese black pine trees, P. thunbergii, has been reported to carry an average of 290 bacterial cells (Guo et al., 2002) on its body surface, which can be observed directly by scanning electron microscopy (Zhao et al., 2000b). A survey of species of bacteria carried by PWN isolated from diseased P. thunbergii and P. massoniana in China indicated that 24 bacterial strains were isolated and identified. Bioassays showed that 17 of the 24 strains could produce toxins, and most of the strains belonged to genus Pseudomonas (Zhao et al., 2003). Since it is a common phenomenon for nematodes to carry bacteria in nature, it is possible that these bacteria may interact with nematodes and contribute to pine wilt to some extent. Therefore, we hypothesize that PWD is a complex, induced by both PWN and the bacteria it carries. To further elucidate the role of bacteria carried by PWN in PWD, we report two phytotoxins, cyclo(-Pro-Val-) and cyclo(-Pro-Tyr-), referred to as compounds 1 and 2, respectively, secreted by P. fluorescens GcM5-1A carried by PWN in vitro, which were toxic to both suspension cells and shoot seedlings of P. thunbergii.