1 Department of Agricultural Biology, Chungbuk National University, Cheongju, 363-763, Korea ([email protected]) Pine wilt is the most important disease of pine trees in Korea, Japan and China. The pathogen causing this disease, the pinewood nematode (Bursaphelenchus xylophylus), is transmitted vectored by adults of some cerambycid beetle species and the Japanese pine sawyer, Monochamus alternatus, is the major vector species in Korea. Although chemical insecticides have been used to kill vector insect and thus prevent transmission of the pathogen, the efficacy is not good. In Japan, to control this insect, an entomopathogenic fungus was studied and developed as an insecticide. This is thought to be the convenient and effective method to control M. alternatus. Recently, there are several reports about the pinewood nematode is vectored by also the pine sawyer, M. saltuarius, in Korea. The objective of this study, therefore, was to isolate and identify entomopathogenic fungi from to control it. We collected M. saltuarius cadaver the cadaver of M. saltuarius and then screened several fungi colonies. The pathogenicity of each fungus was tested using oak longicorn beetle, Moechotypa diphysis, as substitutive insect. M. diphysis is also serious pest to various trees in forest. As the result, only one of them showed high pathogenicity against M. diphysis. Selected fungus was identified by microscopic examination and DNA analysis. Pathogenicity was also evaluated to M. saltuarius 1. lsolation of fungi Fungi were isolated directly from cadavers supporting fungal sporulation, using a semi- selective medium consisting of potato dextrose agar (PDA; Difco TM , USA) containing PENICILLIN- STREPTOMYCIN SOLUTION (Sigma, USA). 2. Culture condition Fungi were grown on potato dextrose agar (PDA; Difco TM , USA) in the dark at 25℃ and store at 4℃ until use. 3. Insects Adults M. saltuarius and Moechotypa diphysis were field-collected and used to bioassay. Fig. 1. Selection of fungus from cadaver of M. saltuarius. Growth of the selected fungus on PDA medium at 25℃ for 3days (A) and 14days (B). Several fungal colonies were isolated from the cadaver and their pathogenicities were assayed against to Moechotypa diphysis for the selection of entomopathogenic fungus. As a result, a kind of fungus was obtained and named as MsW1. Colonies of MsW1 was usually slow growing, downy, at first white but later often becoming yellow to pinkish. Fig. 2. Phase contrast (A) and scanning electron (B) micrographs of MsW1. Conidia and mycelia were mounted from plates and examined under X400 on a Nikon microscope. For the SEM, samples were mounted on stubs, coated with gold and observed in a Carl Zeiss LEO-1530 SEM. MsW1 was characterized by the sympodial development of single-celled conidia (ameroconidia) on a geniculate or zig-zag rachis. 10 8 10 7 10 6 10 5 10 4 Days on 100% mortality conidia/ml Fig. 3. Pathogenicity of MsW1 against to Moechotypa diphysis adults. The pathogenicity was evaluated as till times that mortality reaches to 100%. Conidia of MsW1 obtained from 14-day-old PDA plate were suspended in 0.02% Tween-80 solution. Spores were prepared from 1 × 10 8 conidia/ml to 1 × 10 4 conidia/ml by diirect counting in a haemocytometer. M. diphysis were inoculated by dipping for 10~15 sec into the spore suspension and were maintained in a plant culture chamber at 25℃. Mortality of M. diphysis showed 100% at all concentarions and the pathogenicity increased with increasing conidial concentrations. Fig. 3. Moechotypa diphysis adults infected with MsW1 at 5 days (A) and 10 days (B) after death. The pathogenicity of MsW1 was also confirmed against M. saltuarius adults, but it needs more in detail and repeatedly. P1 5’-AAGCTTCGACATGGTCTG–3’ P3 5‘-GGAGGTGGTGAGGTTCTGTT-3’ 0 524 P5 5‘-AGGAGAGAGCTCGACGGTCA-3’ 333 Fig. 5. Location and sequence of PCR primers for differentiation of the Beauveria spp. Identification of enotomopathogenic fungi was conducted by previous reported primers, P1 (forward), P3 (backward), and P5 (backward) (Dwayne et al. 1996). PCR with the P1-P3 primer set could discriminate isolates of Beauveria spp from other entomopathogenic fungi with a PCR product of about 500bps. The P1-P5 primer set could identify B. bassiana strain specifically from Beauveria spp. with a approximately 330bp within P1-P3 PCR product. M 1 2 600bp 500bp 400bp 300bp 200bp P1-P3 P1-P5 Fig. 6. Agarose gel electrophoresis of P1-P3 and P1-P5 PCR products from MsW1. Genomic DNA was extracted using an I-genomic BYF DNA extraction mini Kit (Intron Co., Korea). The reaction parameters for P1-P3 primer set were as follows; initial denaturation for 3 min at 94 ℃, followed by 35 cycles of 94℃ for 30sec, 53 ℃ for 30sec, and 72 ℃ for 45sec, and a final 10- min extension at 72 ℃. The reaction parameters for P1-P5 primer set were same with those of P1-P3 except the annealing temperature of 50 ℃. Lane M, 100 bp size marker; lane 1, P1-P3 PCR ; lane 2, P1-P5 PCR. The result of PCR with P1-P3 primer set indicated that MsW1 is belonged to Beauveria spp. To discriminate the species of MsW1 more precisely, PCR was performed with the P1-P5 primer set. As the result, a specific PCR product of about 330bp was amplified. This suggests that MsW1 is B. bassiana. Entomopathogenic fungus, MsW1, was isolated from M. saltuarius and was identified with B. bassiana. The pathogenicity of MsW1 against Moechotypa diphysis, as substitutive insect, showed 100% mortality at the tested all concentrations of conidia. We are investigating the potential of MsW1 as a biocontrol agent for M. saltuarius. Dwayne et. al. 1996. J. Invertebr. Pathol. 67:289-299. Mitsuaki Shimazu. 2004. J. Appl. Entomol. 39(3):485-490