3.7.68
RE-EXAMINATION OF THE INDUCED RESISTANCE OF PINE TREES TO PINE WILT DISEASE BY PRIOR INOCULATION WITH AVIRULENT ISOLATES OF THE PINE WOOD NEMATODE

H KOSAKA, T KIYOHARA, T AIKAWA, N OGURA, K TABATA

Forest Biology Division, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan

Background and objectives
Pine wilt disease caused by the pine wood nematode, Bursaphelenchus xylophilus, is a most serious forest disease in Japan [1]. Chemical spray has been a primary procedure to control the disease. Recent public opinion, however, requires new control methods which affect low impact on environment such as biological control. Kiyohara (2] found resistance of pine trees to the disease was induced by prior inoculation with an avirulent isolate of the pine wood nematode. Although this phenomenon has not been remarked particularly, it suggests feasibility to utilize the avirulent isolates as vaccines against the disease. Since Kiyohara's experiments had been conducted on the young pine trees (2-13 year-old) in nurseries, we tried to induce resistance of mature pine trees to pine wilt disease in a countryside forest.

Material and methods
Experiments were conducted on 30 year-old Japanese black pine, Pinus densiflora in a countryside forest at Nanaka Town, central Japan. Avirulent isolates, Cl 4-5 and OKD-1, were used as inducers of the resistance. Virulent isolates, T-4 and Ka-4, were used as challengers. An outline of the methods was as follows. In 1996, 20,000 nematodes of Cl 4-5 and OKD-1 were pre-inoculated to the pine trees on June 3 then inoculated sequentially 10,000 nematodes of T-4 on July 4. In 1997, 50,000 and 20,000 nematodes of OkD-1 were pre-inoculated on June 12 then inoculated sequentially 10,000 nematodes of Ka-4 on July 9. Only the challengers were also inoculated as controls in each year. Inoculated trees were observed in October in the inoculated year and in June in the next year, and were categorized as healthy, infected (alive but showing depressed foliage) or dead.

Result and conclusion
Mortality rates of the trees inoculated with C14-5 + T4, OKD-1 + T4 and T4 alone were 27.3%, 41.7% and 51.2%, respectively, in October 1996; the rates increased, however, to 72.7%, 83.3% and 87.0%, respectively, in June 1997. The reason why mortality rates increased was that the most of infected and some of healthy trees in October turned to the dead in June. In the Kiyohara's experiments conducted on the young pine, the pre-inoculation did not induce complete resistance of the trees and the results similar to our experiments were shown when the numbers of pre-inoculated avirulent nematodes were less than 1 0,000 [2]. He considered the degree of induced resistance might be proportional to inoculum amount of the avirulent nematodes. Accordingly, an avirulent isolate, OkD-i, were pre-inoculated to pine trees with larger amount in 1997 than in 1996. Mortality rates of trees inoculated with 50,000 nematodes of OKD-i + Ka-4, 20,000 OkD-i + Ka-4 and Ka-4 alone were 18.8%, 25% and 60%, respectively, in October 1997; the rates of infected trees were 18.8%, 18.8% and 13.3%, respectively. The inoculated trees will be also observed in June 1998.

In our experiments to date, the delayed symptom development of pine wilt disease was observed definitely. It is, however, unable to say that inoculation with the avirulent nematodes induced adequate resistance of pine trees to the disease because more than 70% of the trees were dead in June 1997. The degree of induced resistance may be different in the tree age and/or in the environmental condition. Pine wilt disease is spread by the way that the adult vector insect, Monochamus alternatus, emerged from dead trees with the nematodes transmits them to healthy trees. The vector can lay eggs only in summer onto the newly dead pine trees. That is, the trees die after autumn and before next summer will not be the sources of next infection. Thus we still consider the pre-inoculation of the avirulent pine wood nematodes is a possible control method of pine wilt disease even if it only delay the symptom development of the disease. We are improving the pre-inoculation method.

References
1. Mamiya Y, 1983. Annual Review of Phytopathology 21, 201-220.
2. Kiyohara T, 1984. Proceedings of the United States-Japan Seminar: The Resistance Mechanisms of Pines Against Pine Wilt Disease, pp.178-184.