lForestry and Forest Products Research Institute, Kukizaki, Ibaraki 305-0903, Japan; 2 Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba City, Ibaraki 305-0006, Japan

Background and objectives
Blue-stain fungi are an important fungal group, together with their associated beetles, because they cause sap-staining in freshly cut logs and some of them have high pathogenicity to trees. The pine shoot beetle Tomicus piniperda (Coleoptera: Scolylidae) is one of the important forest pests on pines in Europe, northern Africa, and Asia including Siberia, and it was also recently discovered in the United States. It usually breeds in dead trees and timber, but may sometimes colonize live, weakened trees. In Europe, Leptographium wingfieldii and Ophiostoma minus are constantly associated with Tomicus piniperda [1]. In addition, the two fungi are considered to have an important role in overcoming the resistance of pine under beetle attack [2]. In Japan, only a few studies have been carried out on the blue-stain fungi on T. piniperda. In this investigation, species composition and isolation frequency of blue-stain fungi during stages in the life cycle of T. piniperda were clarified, and the pathogenicity of isolated fungi was tested.

Materials and methods
Materials were collected from reforested stands (40-45 years old) of Japanese red pine (Pinus densiflora) in Ibaraki, central Japan. Isolation experiments of fungi were conducted from the beetle bodies and its gallery systems at different stages of beetle's life cycle. To test the pathogenicity of isolates, main stems of 33-year-old Japanese red pine were inoculated with 11 fungal species and the length of lesions at the cambial area and the depth of dried zones in the sapwood were measured 6 months after inoculation.

Results and conclusions
Blue-stain fungi were isolated from Tomicus piniperda adults at various developmental stages, its galleries, pupal chambers and sapwood underneath galleries. Among seven species of blue-stain fungi isolated, an Ophiostoma species and O. minus were closely associated with T. piniperda, and were shown to be vectored by the beetle. These two species occurred on newly-emerged adults more frequently than on the overwintered adults that were collected shortly after invading the host tree. The Ophiostoma sp. resembles O. curophioides, but its exact identity remains unclear, because the ascospore size and anamorph morphology are slightly different from this species. Hormonema dematioides, a black yeast, also seemed to be vectored by the beetle. However, its frequency in newly-emergent adults was low. Although the other two species, O. ips and Graphium sp. were also isolated from emergent beetles, their frequency of isolation from gallery systems suggested that they were accidentally carried by T. piniperda. Leptographium wingfieldii was also isolated at a low frequency and this fungus did not seem to be closely associated with the beetle. Ophiostoma sp. and O. minus were considered to be main fungi in the blue-stained area of sapwood of Japanese red pine infested by T. piniperda. In inoculation experiments on Japanese red pine with 11 fungal species, the length of lesions at the cambium area and the depth of dried zones in the sapwood were greatest with Leptographium wingfieldii, followed by the Ophiostorna sp.

1. Gibbs JN, Inman A, 1991. Forestry 64 (3), 239-249.
2. Solheim H, Langstrom B, Hellqvist C, 1993. Canadian Journal of Forest Research 23, 1438-1443.