Nematology, 2006, Vol. 8(1), 59-67

Effect of inoculation on the number of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) carried by alternatus (Coleoptera: Cerambycidae) ∗ Noritoshi MAEHARA 1, ,KakuTSUDA 2,MichimasaYAMASAKI 3, Shinsuke SHIRAKIKAWA 4 and Kazuyoshi FUTAI 4 1 Management Laboratory, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan 2 Gifu Academy of Forest Science and Culture, Gifu 501-3714, Japan 3 Laboratory of Forest Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan 4 Laboratory of Environmental Mycoscience, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan Received: 8 August 2005; revised: 31 October 2005 Accepted for publication: 1 November 2005

Summary – To confirm the effects of -inhabiting fungi on the number of wood (Bursaphelenchus xylophilus) carried by the Japanese pine sawyer (Monochamus alternatus), we attempted to change the mycoflora and also to prevent blue-stain fungi, the main food source fungi of the nematodes, from spreading throughout pine wilt-killed Pinus densiflora wood by inoculating 12 other fungi into the dead logs. Trichoderma sp. 2, Trichoderma sp. 3, and Verticillium sp. inoculation treatments tended to decrease the number of the nematodes carried by the beetles. These fungi were more or less antagonistic to the blue-stain fungus Ophiostoma minus,andTrichoderma sp. 2 and Trichoderma sp. 3 grew faster than O. minus on four kinds of media. Keywords – blue-stain fungi, Japanese pine sawyer, pine wood , Trichoderma spp., Verticillium sp.

The pine wood nematode, Bursaphelenchus xylophilus chamus beetles among individual pine trees from which (Steiner & Buhrer) Nickle, the causal agent of pine wilt the beetles emerged (Maehara et al., 2005). Another im- disease (Kiyohara & Tokushige, 1971), is vectored from portant factor which affects the number of nematodes car- wilt-killed to healthy by the Japanese pine sawyer, ried by beetles is the fungal flora in the wood. In vitro Monochamus alternatus Hope in Japan (Mamiya & Enda, studies showed that beetles carried a greater number of 1972; Morimoto & Iwasaki, 1972). The number of nema- nematodes when the blue-stain fungus Ophiostoma mi- todes that enter a healthy tree is directly proportional to nus (Hedgcock) H. & P. Sydow was dominant in wood the number of nematodes carried by Monochamus beetles around artificial pupal chambers of the beetles, while (Togashi, 1985). The rate of disease development is di- the numbers of nematodes decreased when Trichoderma rectly related to the number of nematodes in the inoculum spp. or Verticillium sp. was prevalent (Maehara & Fu- (Kiyohara et al., 1973). Thus, to understand the dynamics tai, 1996, 1997). The former fungus is suitable and the of pine wilt disease development, it is important to iden- latter two fungi are unsuitable for nematode propagation tify the factors affecting the number of nematodes carried (Fukushige, 1991; Maehara & Futai, 2000). In field sur- by an individual beetle, which ranges from zero to over veys, intense blue-stain on the pupal chamber walls of 200 000. M. alternatus increased the number of nematodes aggre- Previous studies showed that M. alternatus emerging gating around such chambers and the number carried by from extremely dry or wet pupal chambers (Morimoto & the beetles which emerged from the chambers (Maehara et Iwasaki, 1973; Maehara et al., 2005), or logs (Terashita, al., 2005). In the present study, to confirm effects of wood- 1975; Kobayashi et al., 1976; Togashi, 1989; Fukushige, inhabiting fungi on the number of nematodes carried by 1990) carried relatively few nematodes. There were dif- the beetles, we attempted to change the mycoflora and ferences in the numbers of nematodes carried by Mono- also to prevent blue-stain fungi from spreading through-

∗ Corresponding author, e-mail: [email protected]

© Koninklijke Brill NV, Leiden, 2006 59 Also available online - www.brill.nl

Downloaded from Brill.com09/23/2021 03:10:02PM via free access N. Maehara et al. out pine wilt-killed Japanese red pine (Pinus densiflora lation, each log was placed lengthwise in a plastic con- Sieb. & Zucc.) wood by inoculating 12 other fungi into tainer (25 cm diam. × 28 cm deep), then covered with a the dead logs. steel mesh. The containers were placed in a shed at Ki- tashirakawa Experimental Station, Field Science Educa- tion and Research Center, Kyoto University, Kyoto City, Materials and methods Kyoto Prefecture. The logs were watered weekly during the experiment. They were observed daily from June to INOCULATION OF FUNGI INTO PINE TREES KILLED BY July 1991. PINE WILT IN 1990 INOCULATION OF FUNGI INTO PINE TREES KILLED BY Four 30- to 40-year-old P. densiflora trees, killed by PINE WILT IN 1992 pine wilt in 1990, were felled at Kamigamo Experimen- tal Station, Field Science Education and Research Center, Six 30 to 40-year-old P. densiflora trees, killed by wilt Kyoto University, Kyoto City, Kyoto Prefecture, Japan, on in 1992, were felled at Kamigamo Experimental Station 14 November 1990. Eighty logs (25 cm long, ca 10 cm in on 22 October 1992. One hundred and four logs (25 cm mid-diam.) were cut from the trees. All the trees had lar- long, 7.4-14.8 cm in mid-diam.) were cut from the trees. vae of M. alternatus. Not all the logs, however, had them. All the trees had larvae of M. alternatus. Not all the logs, The following day, each log was inoculated with 7 mm however, had them. Five hundred beech wood chips (each diam. mycelial disks of one of the test fungi growing on 20 mm long × 9 mm diam.) were autoclaved with 100 ml potato dextrose agar (PDA). Inoculation was made via two distilled water for 25 min. The chips were placed on one drill holes (each 11 mm diam. × ca 20 mm deep), one hole of the test fungi growing on PDA and cultured at 20◦C at each cut end and the opposite side of each log, then each for 25 days. On 23 October 1992, each log was inoculated hole was plugged with a rubber stopper. Ten of with the chips of one of the test fungi. The reason why fungi were inoculated: Arthrobotrys sp., Coriolus hirsu- we did not use PDA disks but wood chips with fungi in tus (Wulf.: Fr.) Quél., Cryptoporus volvatus (Peck) Shear, this year was that PDA dries faster than the wood chips. Cystidiophorus castaneus (Lloyd) Imaz., Gloeophyllum Inoculation was made through two drill holes, 9 mm diam. striatum (Swartz: Fr.) Murrill, Pycnoporus coccineus (Fr.) × ca 20 mm deep: one hole at each cut end and opposite Bond. & Sing., Trichaptum abietinum (Dicks.: Fr.) Ryv., side of each log. Fungi inoculated were Trichoderma Trichoderma sp. 2, Trichoderma sp. 3, and an unidentified sp. 2, Trichoderma sp. 3, P. coccineus, Pleurotus ostreatus species of Basidiomycetes (Table 1). These fungi could (Jacq.: Fr.) Kummer, and Verticillium sp. (Table 1). be isolated from dead pine trees. The logs which served Pleurotus ostreatus could be also isolated from dead pine as controls were not inoculated with any fungi and the trees. Further inoculations with Trichoderma sp. 3 were holes were filled with rubber stoppers. After the inocu- also carried out by boring another two or four holes in the

Table 1. Origin of fungi used. Fungus Origin and reference Arthrobotrys sp. Pine wood nematode-inoculated Pinus thunbergii (Fukushige & Futai, 1987) Coriolus hirsutus Isolated from pine trees by H. Fukushige Cryptoporus volvatus Forestry and Forest Products Research Institute Cystidiophorus castaneus Forestry and Forest Products Research Institute Gloeophyllum striatum Forestry and Forest Products Research Institute Ophiostoma minus Pine wood nematode-infected Pinus densiflora (Fukushige, 1991) Pleurotus ostreatus Kyoto Prefectural Forestry Experimental Station Pycnoporus coccineus Forestry and Forest Products Research Institute Trichaptum abietinum Forestry and Forest Products Research Institute Trichoderma sp. 2 Pine wood nematode-inoculated P. thunbergii (Fukushige & Futai, 1987) Trichoderma sp. 3 Pine wood nematode-infected P. densiflora (Fukushige, 1991) Verticillium sp. Pine wood nematode in the nematode-infected P. densiflora (Fukushige, 1991) Unidentified species of Basidiomycetes Isolated from pine trees by H. Fukushige

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Downloaded from Brill.com09/23/2021 03:10:02PM via free access Effect of fungus inoculation on the number of Bursaphelenchus xylophilus carried by Monochamus alternatus side of each log (four or six points in total). Logs serving They were grown on PDA, 1/10 strength PDA, 1.5% wa- as controls were inoculated with two axenic wood chips. ter agar, and 1.5% water agar supplemented with 0.5% After the inoculation with fungi, each log was placed dry sawdust (w/w) of Japanese black pine (Pinus thun- lengthwise in a plastic container, 25 cm diam. × 28 cm bergii Parl.). Ophiostoma minus and one of the six test deep. The containers were placed in the same shed as in fungi were co-cultured on the 15 ml test media in 9 cm 1990, and each was covered with a steel mesh on 21 May diam. Petri dishes. For inoculum, mycelial disks, 7 mm 1993. The logs were watered at least once a week during diam., were cut from the margin of fungus colonies grow- the experiment. They were observed daily from June to ingonPDA.AdiskofO. minus and another disk of the August 1993. other fungi were co-inoculated, 3 cm opposite one another on the centre of a dish. Control cultures received only one NUMBER OF NEMATODES CARRIED BY MONOCHAMUS disk of each fungus at 3 cm from the centre of the dish. ALTERNATUS Each treatment was replicated five times. The inoculated dishes were incubated at 20◦C in the dark and growth Upon emergence of M. alternatus from the logs, each of each fungus colony was measured on a line through beetle was ground for 10 s in 40 ml tap water and the the centres of the two colonies each day for Trichoderma suspension was placed in a Baermann funnel overnight to sp. 2, Trichoderma sp. 3 and Arthrobotrys sp. and every extract the nematodes in 1991 and 1993. These were then second day for Verticillium sp., P. ostreatus and P. coc- counted using a stereomicroscope. When the nematodes cineus for 30 days. were too abundant for counting, the suspension was diluted and the number of nematodes was estimated. STATISTICAL ANALYSIS

GROWTH OF TRICHODERMA IN WOOD One-way analysis of variance (ANOVA) was used to analyse the differences in the numbers of nematodes To investigate the growth of Trichoderma sp. 3 in among individual trees or fungus treatments. For ANOVA, wood, a 35-year-old P. densiflora tree, killed by wilt the numbers of nematodes were log10-transformed. A in 1992, was felled at Kamigamo Experimental Station chi-square test was used to determine if the percentage on 10 November 1992. Seven logs (25 cm long, 12.9- of M. alternatus carrying less than 100 nematodes was 14.7 cm in mid-diam.) were cut from the tree. Each dependent on fungus treatments. log was inoculated with two wood chips of Trichoderma sp. 3 in the same way as mentioned above and placed lengthwise in a plastic container, 25 cm diam. × 28 cm Results deep. Monthly from December 1992 to June 1993, a log was cut vertically with a band saw and the distribution of EFFECT OF FUNGUS INOCULATION ON THE NUMBER fungi on the surface of vertical section was determined. OF NEMATODES CARRIED BY MONOCHAMUS At each sampling date, 11 wood chips were hollowed out ALTERNATUS evenly from the surface with a 10 mm diam. punch, flame- sterilised, then placed on PDA. After incubation at 20◦C From 20 June-11 July 1991, 101 Monochamus beetles for 1 month, the number of chips from which Trichoderma emerged. The numbers of nematodes (fourth-stage disper- spp., including Trichoderma sp. 3, grew was determined. sal juveniles: J4) carried by the individual beetle ranged In June 1993, a control log inoculated with two axenic from zero to 85 500, the mean being 4585. The beetles that wood chips (on 23 October 1992) was also cut in the same emerged from Tree 1 carried fewer J4, although there was way as the log inoculated with Trichoderma sp. 3. no significant difference (P = 0.289) (Table 2). There- fore, we analysed the effect of fungus inoculation on the ANTAGONISM OF SEVERAL FUNGI AGAINST numbers of J4 carried by a beetle with the exception of OPHIOSTOMA MINUS the data from Tree 1 (Table 3). One beetle only emerged from the logs inoculated with C. hirsutus and the number The fungi used were the blue-stain fungus Ophiostoma of J4 carried by the beetle was one. The datum was not in- minus (called Ceratocystis sp. 2 by Fukushige (1991)) cluded in Table 3 because the beetle emerged from Tree 1. (Table 1), Trichoderma sp. 2, Trichoderma sp. 3, Verticil- Trichoderma sp. 2, P. coccineus, and Trichoderma sp. 3 lium sp., Arthrobotrys sp., P. ostreatus and P. coccineus. treatments tended to decrease the number of J4 carried

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Table 2. Numbers of pine wood nematodes carried by Monochamus alternatus which emerged from each tree of pine wilt-killed Pinus densiflora in 1991. Tree number Number of beetles Number of J4 carried by a beetle* 1739.6 ± 71.7 2 38 5717.9 ± 13178.3 3 9 9875.4 ± 28363.3 4 47 3333.0 ± 5082.2

* Values are means ± SD.

Table 3. Effect of inoculating pine wilt-killed Pinus densiflora logs with fungi on the numbers of pine wood nematodes carried by emerging Monochamus alternatus in 1991.

Fungus treatment Number of Number of Number of J4 carried by a beetle* Percentage of the beetles logs beetles carrying fewer than 100 nematodes Trichoderma sp. 2 5 5 299.6 ± 648.9 80.0 Pycnoporus coccineus 5 10 1205.9 ± 2784.9 60.0 Trichoderma sp. 3 6 10 2204.5 ± 5609.4 80.0 Cystidiophorus castaneus 5 6 3255.2 ± 2860.6 16.7 Unidentified species of Basidiomycetes 6 15 4353.3 ± 12519.9 60.0 Trichaptum abietinum 6 12 5044.8 ± 6591.9 41.7 Arthrobotrys sp. 5 9 7473.8 ± 15709.0 44.4 Gloeophyllum striatum 4 12 10224.7 ± 24144.1 33.3 Cryptoporus volvatus 5 4 12063.5 ± 24091.0 75.0 Control (no fungus) 7 11 3965.8 ± 5167.4 36.4

* Values are means ± SD.

Table 4. Numbers of pine wood nematodes carried by Monochamus alternatus which emerged from each tree of pine wilt-killed Pinus densiflora in 1993. Tree number Number of beetles Number of J4 carried by a beetle* 1 15 498.3 ± 856.4 2 11 400.3 ± 800.7 3 29 401.9 ± 1659.9 4 45 2490.2 ± 5018.5 52346.3 ± 50.0 6 12 207.8 ± 545.0

* Values are means ± SD. by a beetle; however, the differences were not significant From 21 June-5 August 1993, 135 Monochamus beetles (P = 0.219) because of great variance in the numbers emerged. The numbers of J4 carried by the individual of J4 among individual beetles. The percentage of beetles beetle ranged from zero to 22 600, the mean being carrying fewer than 100 nematodes also tended to be high 1037.3. The beetles that emerged from Tree 5 carried in the logs inoculated with Trichoderma sp. 2 and Tricho- fewer J4 than those from the other trees (P<0.0001) derma sp. 3 (P = 0.180). By contrast, the number of J4 (Table 4). Therefore, we analysed the effect of fungus carried by a beetle from the logs inoculated with Arthro- inoculation on the numbers of J4 carried by a beetle botrys sp. and the Basidiomycetes except P.coccineus was with the exception of the data from Tree 5 (Table 5). similar to or higher than that in the controls. As in the 1991 result, the number of J4 carried by

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Table 5. Effect of inoculating pine wilt-killed Pinus densiflora logs with fungi on the numbers of pine wood nematodes carried by emerging Monochamus alternatus in 1993.

Fungus treatment Number of Number of Number of J4 carried by a beetle* Percentage of the beetles logs beetles carrying fewer than 100 nematodes Trichoderma sp. 3 (four inoculation points/log) 10 11 333.5 ± 408.0 45.5 Verticillium sp. 11 15 442.8 ± 644.1 46.7 Trichoderma sp. 2 13 16 687.2 ± 1232.4 56.3 Trichoderma sp. 3 (six inoculation points/log) 11 16 700.6 ± 1841.8 68.8 Pleurotus ostreatus 11 11 736.4 ± 1489.0 45.5 Pycnoporus coccineus 11 20 1085.0 ±1911.9 35.0 Trichoderma sp. 3 (two inoculation points/log) 9 12 2723.6 ±6201.9 33.3 Control (no fungus) 10 11 3917.1 ±7695.8 36.4

* Values are means ± SD. a beetle varied even when beetles emerged from the logs receiving the same fungus. Therefore, there was no significant difference among fungus treatments (P = 0.649). However, the Trichoderma sp. 3 (four- and six- point inoculation), Verticillium sp., and Trichoderma sp. 2 treatments seemed to decrease the number of J4 carried by a beetle. The percentage of beetles carrying less than 100 nematodes also seemed to be high in the logs inoculated with Trichoderma sp. 3 (six-point inoculation), although there was no significant difference (P = 0.521).

GROWTH OF TRICHODERMA IN WOOD

Isolation frequency of Trichoderma spp. increased over time (Fig. 1). Almost all Trichoderma spp. were Trichoderma sp. 3, although we could not judge whether the rest of them were Trichoderma sp. 3 or not. Seven months after inoculation with Trichoderma sp.3,the Fig. 1. Isolation frequency of Trichoderma spp. including Tri- spread of blue-stain was limited in the logs inoculated choderma sp. 3 from wilt-killed Pinus densiflora logs inoculated with Trichoderma sp. 3 in comparison with the control log with Trichoderma sp. 3. (Fig. 2A, B). The suppression of blue-stain in the control log in Figure 2B must be attributed to a gnarl in the log. than on the other media. Verticillium sp. was the slowest ANTAGONISM OF SEVERAL FUNGI AGAINST growing fungus on all media. OPHIOSTOMA MINUS Table 6 shows the interactions between O. minus and the other fungi. All the fungi prevented the growth of Figure 3 shows the average growth of the seven O. minus on all media. Even after contacting O. minus, fungi monocultured on PDA, 1/10 strength PDA, water Trichoderma sp. 2 and Trichoderma sp. 3 continued to agar, and water agar supplemented with pine sawdust. grow up to the edge of each Petri dish on all media. Trichoderma sp. 2 and Trichoderma sp. 3 grew faster than Verticillium sp. colonies failed to reach the edge of all other fungi on each medium. The next fastest grower the dishes during experimental period. The growth of was O. minus, but its growth was slow on water agar. the other fungi depended on the test medium, e.g., By contrast, Arthrobotrys sp. grew faster on water agar Arthrobotrys sp. could cover the dishes on all media

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except PDA. Thus, all six species of fungi were more or less antagonistic to O. minus.

Discussion

Kobayashi et al. (1974, 1975), Fukushige and Futai (1987), Wingfield (1987), and Maehara et al. (2005) iso- lated blue-stain fungi at higher frequency from nematode- infested or nematode-inoculated pine trees. Kobayashi et al. (1974, 1975), Fukushige (1991), and Maehara and Futai (2000) compared nematode propagation on vari- ous fungi isolated from healthy and wilt-killed pines and showed that, while the nematodes fed and multiplied on some of these fungi, others were unsuitable for nematode propagation. Blue-stain fungi were extremely suitable for the nematode propagation. Fukushige (1990) found a pos- itive relationship between the degree of blue-stain and the density of the nematodes in dead wood of Korean pine (Pinus koraiensis Sieb. & Zucc.). Fukushige (1991) and Maehara and Futai (2000) also reported that blue-stain fungi were highly suitable for the occurrence of third- stage dispersal juveniles (J3) of the nematodes because a high population density is a prerequisite for the oc- currence of J3. J3 moults to J4 in the presence of insect vectors of the Monochamus (Morimoto & Iwasaki, 1973; Warren & Linit, 1993; Maehara & Futai, 1996; Necibi & Linit, 1998; Maehara & Futai, 2001), and then Fig. 2. Vertical sections of wilt-killed Pinus densiflora logs J4 transfers from infested wood to vectors. inoculated with A: Trichoderma sp. 3; and B: with no fungus In vitro studies showed that when the blue-stain fun- (control) 7 months after fungus inoculation. gus O. minus dominated wood around artificial pupal

Table 6. Types of interactions observed between Ophiostoma minus and other fungi.

Fungus treatment Number of PDA 1/10 strength Water agar Water agar supplemented observations PDA with Pinus thunbergii sawdust Trichoderma sp. 2 5 A (17.6) C (20.8) C (17.5) C (30.3) Trichoderma sp. 3 5 A (13.5) C (19.4) C (21.9) C (30.5) Arthrobotrys sp. 5 E (43.4) C (34.2) C (22.8) C (32.0) Verticillium sp. 5 B (49.3) D (47.6) E (32.4) D (38.3) Pleurotus ostreatus 5 A (44.8) C (42.8) C (20.3) D (38.1) Pycnoporus coccineus 5 B (42.0) D (35.7) C (28.9) D (36.2) A: Challenger grew over Ophiostoma minus; O. minus growth ceased. B: Challenger grew over O. minus slightly; O. minus growth ceased. C: Hyphae of O. minus and challenger intermingled and challenger grew up to the edge; O. minus growth ceased. D: Hyphae of O. minus and challenger intermingled and challenger grew slightly; O. minus growth ceased. E: Hyphae of O. minus and challenger intermingled and growth of both ceased. The numbers in brackets indicate the final growth (mm) of O. minus measured as the distance from the initial margin of the inoculum to the final margin of the growing colony.

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Fig. 3. Growth of fungi monocultured on PDA, 1/10 strength PDA, water agar, and water agar supplemented with Pinus thunbergii sawdust. Tri2, Trichoderma sp. 2; Tri3, Trichoderma sp. 3; Ver, Verticillium sp.; Ple, Pleurotus ostreatus;Art,Arthrobotrys sp.; Pyc, Pycnoporus coccineus; Oph, Ophiostoma minus. Values are means of five observations. chambers of M. alternatus, the beetles emerging from source fungi of the nematodes in the wood should reduce the chambers carried many nematodes (Maehara & Futai, the density of the nematodes, and in turn reduce the 1996, 1997). In field surveys, intense blue-stain of the pu- number of J4 carried by a beetle. Trichoderma sp. 2, pal chamber walls in wilt-killed pine trees also increased Trichoderma sp. 3, and Verticillium sp. treatments tended the numbers of J4 carried by the beetles which emerged to decrease the number of J4 carried by a beetle. In from the chambers, i.e., blue-stain fungi greatly affected vitro studies also showed that the numbers of nematodes the number of J4 carried by the beetles (Maehara et al., carried by a beetle decreased when these three fungi 2005). Blue-stain fungi also affected the differences in dominated wood around artificial pupal chambers of the number of J4 carried by several other species of - the beetles (Maehara & Futai, 1996, 1997). Fukushige tles which emerged from wilt-killed pine trees (Maehara (1991) and Maehara and Futai (2000) reported that & Futai, 2002). the nematode population on Verticillium sp. decreased To confirm the effects of fungi on the number of drastically because this fungus is a nematode parasite, J4 carried by M. alternatus, we changed the mycoflora and that Trichoderma sp. 2 and Trichoderma sp. 3 were composition and also prevented blue-stain fungi from not suitable for the nematode propagation. In the present spreading throughout killed pine wood by inoculating study, these fungi, especially Trichoderma sp. 2 and other fungi into the dead logs. Eradicating the main food- Trichoderma sp. 3 which also grew faster than O. minus,

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Downloaded from Brill.com09/23/2021 03:10:02PM via free access N. Maehara et al. were antagonistic to O. minus, although the results varied of the 27th annual meeting of Kyushu Branch of the Japanese depending upon the media used. Moreover, Trichoderma Forestry Society, 161. sp. 3 suppressed the spread of blue-stain in the logs. This FUKUSHIGE, H. (1990). The number of Bursaphelenchus xy- resulted in the decrease of the number of J4 carried by a lophilus carried by Monochamus alternatus and some possi- beetle by the inoculation of these fungi into the logs. ble factors regulating the number. Japanese Journal of Nema- Pleurotus ostreatus immobilises and digests nematodes tology 20, 18-24. (Thorn & Barron, 1984; Barron & Thorn, 1987), and FUKUSHIGE, H. (1991). Propagation of Bursaphelenchus xy- lophilus (Nematoda: Aphelenchoididae) on fungi growing in also preys on pine wood nematodes (Mamiya et al., pine-shoot segments. Applied Entomology and Zoology 26, 2005), and so is unsuitable for the propagation of the 371-376. nematodes on PDA (Dozono, 1974). Mamiya et al. FUKUSHIGE,H.&FUTAI, K. (1987). Seasonal changes in (2005) also reported that T. abietinum and C. volvatus Bursaphelenchus xylophilus populations and occurrence of had a limited ability to prey on pine wood nematodes. fungi in Pinus thunbergii trees inoculated with the nematode. Saiki et al. (1984) tried to control the nematodes in Japanese Journal of Nematology 17, 8-16. P. densiflora and P. thunbergii seedlings by spraying KIYOHARA,T.&TOKUSHIGE, Y. (1971). [Inoculation exper- spores of Arthrobotrys sp., a nematode-trapping fungus, iments of a nematode, Bursaphelenchus sp., onto pine trees.] and confirmed the limited effect of the fungus on the Journal of Japanese Forestry Society 53, 210-218. nematodes. In the present study, there were, however, no KIYOHARA,T.,DOZONO,Y.,HASHIMOTO,H.&ONO,K. clear effects of treatments of these fungi on the number of (1973). [Correlation between number of inoculated nema- J4 carried by a beetle. Therefore, it is suggested that these todes and disease occurrence in pine wilt disease.] Transac- fungi may not spread well in the logs. tions of the 26th annual meeting of Kyushu Branch of the Microbial control of nematodes by fungi may be Japanese Forestry Society, 191-192. KOBAYASHI,T.,SASAKI,K.&MAMIYA, Y. (1974). [Fungi successful when interactions of the nematodes, wood- associated with Bursaphelenchus lignicolus, the pine wood inhabiting fungi, and the beetles are better understood. nematode (I).] Journal of Japanese Forestry Society 56, 136- 145. Acknowledgements KOBAYASHI,T.,SASAKI,K.&MAMIYA, Y. (1975). [Fungi associated with Bursaphelenchus lignicolus, the pine wood nematode (II).] Journal of Japanese Forestry Society 57, 184- We sincerely thank Dr J.R. Sutherland, Consultant, 193. Canada, for critically reviewing an earlier version of KOBAYASHI,K.,OKUDA,M.&HOSODA, R. (1976). [Influ- the manuscript. We are grateful to Kyoto Prefectural ence of wood size and humidity on the emergence of Japanese Forestry Experimental Station and Mori & Company Ltd pine sawyer and the number of pine wood nematodes per in- for providing us with P. ostreatus and beech wood chips, sect.] Transactions of the 87th annual meeting of the Japanese respectively, and to Dr T. Hattori, Forestry and Forest Forestry Society, 239-240. Products Research Institute, for his advice on the MAEHARA,N.&FUTAI, K. (1996). Factors affecting both of wood-decaying basidiomycetes. We are indebted to the the numbers of the pinewood nematode, Bursaphelenchus staff of Kamigamo Experimental Station, Field Science xylophilus (Nematoda: Aphelenchoididae), carried by the Japanese pine sawyer, Monochamus alternatus (Coleoptera: Education and Research Center, Kyoto University for Cerambycidae), and the nematode’s life history. Applied their assistance in collecting and cutting dead pine trees, Entomology and Zoology 31, 443-452. and to the staff of Kitashirakawa Experimental Station, MAEHARA,N.&FUTAI, K. (1997). Effect of fungal inter- Field Science Education and Research Center, Kyoto actions on the numbers of the pinewood nematode, Bur- University for permission to use the shed. saphelenchus xylophilus (Nematoda: Aphelenchoididae), car- ried by the Japanese pine sawyer, Monochamus alternatus (Coleoptera: Cerambycidae). Fundamental and Applied Ne- References matology 20, 611-617. MAEHARA,N.&FUTAI, K. (2000). Population changes of BARRON,G.L.Þ, R.G. (1987). Destruction of nema- the pinewood nematode, Bursaphelenchus xylophilus (Nema- todes by species of Pleurotus. Canadian Journal of Botany, toda: Aphelenchoididae), on fungi growing in pine-branch 65, 774-778. segments. Applied Entomology and Zoology 35, 413-417. DOZONO, Y. (1974). [Reproduction of Bursaphelenchus ligni- MAEHARA,N.&FUTAI, K. (2001). Presence of the ceramby- colus on the colony of various kinds of fungi.] Transactions cid beetles Psacothea hilaris and Monochamus alternatus af-

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fecting the life cycle strategy of Bursaphelenchus xylophilus. SAIKI,H.,SAITO,T.,YONEDA,K.,KAIJO,M.,UCHIDA,K. Nematology 3, 455-461. &YAMANAKA, K. (1984). Biological control of the pine- MAEHARA,N.&FUTAI, K. (2002). Factors affecting the num- wood nematode by spraying a nematode-trapping fungus. ber of Bursaphelenchus xylophilus (Nematoda: Aphelenchoi- Journal of Japanese Forestry Society 66, 30-32. didae) carried by several species of beetles. Nematology 4, TERASHITA, T. (1975). [Relations between water content of 653-658. wood, Bursaphelenchus lignicolus density in wood and the MAEHARA,N.,HATA,K.&FUTAI, K. (2005). Effect of blue- numbers of the nematode carried by beetles emerged from stain fungi on the number of Bursaphelenchus xylophilus (Ne- diseased trees.] Transactions of the 26th annual meeting of matoda: Aphelenchoididae) carried by Monochamus alterna- Kansai Branch of the Japanese Forestry Society, 279-281. tus (Coleoptera: Cerambycidae). Nematology 7, 161-167. THORN,R.G.&BARRON, G.L. (1984). Carnivorous mush- MAMIYA,Y.&ENDA, N. (1972). Transmission of Bursaphe- lenchus lignicolus (Nematoda: Aphelenchoididae) by Mono- rooms. Science, 224, 76-78. chamus alternatus (Coleoptera: Cerambycidae). Nematolo- TOGASHI, K. (1985). Transmission curves of Bursaphelenchus gica 18, 159-162. xylophilus (Nematoda: Aphelenchoididae) from its vector, MAMIYA,Y.,HIRATSUKA,M.&MURATA, M. (2005). Ability Monochamus alternatus (Coleoptera: Cerambycidae), to pine of wood-decay fungi to prey on the pinewood nematode, trees with reference to population performance. Applied Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle. Entomology and Zoology 20, 246-251. Japanese Journal of Nematology 35, 21-30. TOGASHI, K. (1989). Factors affecting the number of Bur- MORIMOTO,K.&IWASAKI, A. (1972). [Role of Mono- saphelenchus xylophilus (Nematoda: Aphelenchoididae) car- chamus alternatus (Coleoptera: Cerambycidae) as a vector of ried by newly emerged adults of Monochamus alternatus Bursaphelenchus lignicolus (Nematoda: Aphelenchoididae).] (Coleoptera: Cerambycidae). Applied Entomology and Zool- Journal of Japanese Forestry Society 54, 177-183. ogy 24, 379-386. MORIMOTO,K.&IWASAKI, A. (1973). [Studies on the pine WARREN,J.&LINIT, M.J. (1993). Effect of Monochamus sawyer (IV). Biology of the pine sawyer and the pine wood carolinensis on the life history of the pinewood nematode, nematode in the pupal cell.] Transactions of the 26th annual Bursaphelenchus xylophilus. Journal of Nematology 25, 703- meeting of Kyushu Branch of the Japanese Forestry Society, 199-200. 709. NECIBI,S.&LINIT, M.J. (1998). Effect of Monochamus WINGFIELD, M.J. (1987). Fungi associated with the pine wood carolinensis on Bursaphelenchus xylophilus dispersal stage nematode, Bursaphelenchus xylophilus, and cerambycid bee- formation. Journal of Nematology 30, 246-254. tles in Wisconsin. Mycologia 79, 325-328.

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