Appl. Entomol. Zool. 41 (1): 123–128 (2006)

Appl. Entomol. Zool. 41 (1): 123–128 (2006) http://odokon.ac.affrc.go.jp/

Death of Quercus crispula by inoculation with adult Platypus quercivorus (Coleoptera: Platypodidae)

Haruo KINUURA1,* and Masahide KOBAYASHI2 1 Kansai Research Center, Forestry and Forest Products Research Institute; Kyoto 612–0855, Japan 2 Kyoto Prefectural Forestry Experimental Station; Kyoto 629–1121, Japan (Received 10 December 2004; Accepted 27 October 2005)

Abstract Adult Platypus quercivorus beetles were artificially inoculated into Japanese oak trees (Quercus crispula). Two inoculation methods were used: uniform inoculation through pipette tips, and random inoculation by release into netting. Four of the five trees that were inoculated uniformly died, as did all five trees that were inoculated at random. Seven of the nine dead trees showed the same wilting symptoms seen in the current mass mortality of oak trees. Raffaelea quercivora, which has been confirmed to be the pathogenic fungus that causes wilt disease and is usually isolated from the mycangia of P. quercivorus, was isolated from all of the inoculated dying trees. Trees that died faster showed a higher density of beetle galleries that succeeded in producing offspring. We found positive relationships between the density of beetle galleries that succeeded in producing offspring and the rate of discoloration in the sapwood and the isolation rate of R. quercivora. Therefore, we clearly demonstrated that P. quercivorus is a vector of R. quercivora, and that the mass mortality of Japanese oak trees is caused by mass attacks of P. quercivorus.

Key words: Mass mortality; oak; pathogenic fungi; Raffaelea quercivora; vector

An inoculation test on some smaller trees in the INTRODUCTION nursery with this fungus, which was later described In Japan, the mass mortality of oak trees (Quer- as Raffaelea quercivora Kubono et Shin. Ito cus crispula BlumeQ. mongolica Fisch. var. (Kubono and Ito, 2002) strongly suggested that this grosserrata Rehd. et Wils. and Q. serrata Thunb. fungus might be responsible for the oak tree die-off ex Murray) has been reported in several prefectures (Ito et al., 1998). These results suggest that R. along the Sea of Japan, in the southern part of quercivora is transferred to oak trees by P. quer- Kyushu Island and on the Kii Peninsula since the civorus and plays a causal role in the mass mortal- latter half of the 1980s (Kinuura, 1994; Ito and Ya- ity of oak trees in Japan. mada, 1998; Ito, 2002). Numerous tunnels of the However, these results alone are not sufﬁcient to ambrosia beetle Platypus quercivorus (Murayama) conclude that an insect is a vector of a plant were observed in all dead and dying trees, and an pathogen. The following four requirements must be unknown species of moniliaceous fungus was iso- met to demonstrate this relationship unequivocally lated predominantly from discolored sapwood, bee- (Leach, 1940). A close, although not necessarily tle galleries in dead oak trees, and the body sur- constant, association of the insect with the diseased faces and mycangia of P. quercivorus (Ito et al., plants must be demonstrated; it must be shown that 1998; Kinuura, 2002). Platypus quercivorus, which the insect also regularly visits healthy plants under belongs to the family Platypodidae, bores into conditions suitable for transmission of the disease; stems and constructs a gallery called a pinhole in the presence of the pathogen or virus in or on the the sapwood. This gallery branches several times insect in nature or following a visitation to a dis- laterally and vertically. Males initiate galleries, and eased plant must be conﬁrmed; and the disease a single female joins each male. must be induced experimentally as a result of in-

*To whom correspondence should be addressed at: E-mail: [email protected] DOI: 10.1303/aez.2006.123

123 124 H. KINUURA and M. KOBAYASHI sect visitation under controlled conditions with ad- were coated with paraffin paste to prevent dehydra- equate checks. tion. Each billet was placed in an unglazed flower- Regarding the relationship between P. quer- pot covered with a black rubber sheet, to which a civorus and Japanese oak wilt disease, mass infes- PET bottle and a PE bottle containing wet tissue tations of this species have been observed among paper were attached to collect dispersing adult P. dying and dead trees, and wilting symptoms be- quercivorus (Kobayashi et al., 2002). Adult beetles come evident following such attacks (Kinuura, were found in the PE bottles almost every morning 1994; Urano, 2000). Therefore, the first two re- from mid-June to late July 2003. These beetles quirements have been met. In addition, R. quer- were sorted by sex and held in plastic containers civora have been isolated from beetle galleries in with wet tissue paper at a constant temperature of dead trees as well as from the proventriculus and 15°C to prevent further wandering. mycangia of P. quercivorus (Kinuura, 2002), meet- Method of inoculation. In late June 2003, 15 ing the third requirement. However, wilting symp- apparently healthy Q. crispula (ca. 10 cm DBH) toms have not been reproduced by inoculation of P. that had not been attacked by P. quercivorus were quercivorus into healthy trees, although Q. crispula selected, because in the current mass mortality of that were greatly stressed by debarking were inocu- oak trees, Q. crispula over 8 cm DBH are generally lated and death was induced (Kinuura, 2005). attacked (Kinuura, 1994). The 15 selected trees Therefore, the fourth requirement has not yet been were covered with a vinyl seal from the stem base met, and it is not clear whether other agents be- to a height of about 4 m to avoid attacks by natural sides P. quercivorus could cause wilting death in Q. P. quercivorus populations (Kobayashi et al., crispula before beetle infestations. The purpose of 2001). In addition, creosote was spread on the root this study was to induce the disease experimentally system to prevent infestations to the stem base. by inoculating P. quercivorus into Q. crispula and Part of the vinyl seal at a height of 50–70 cm was to demonstrate that mass mortality of oak trees in then stripped off with a knife to expose the bark. Japan is caused by concentrated injury from P. These pre-inoculation processes were conducted in quercivorus. late June 2003. Two inoculation methods were used: uniform, compulsory inoculation via pipette tips; and ran- MATERIALS AND METHODS dom, free inoculation by releasing beetles into net- Study area and collection of beetles for inocu- ting surrounding a tree. Each inoculation method lation. This study was conducted in a secondary was applied to five trees, and an additional five natural broad-leaved forest in central Kyoto Prefec- trees were monitored as controls. ture (Keihoku Town, N35°14, E135°38). The a. Uniform inoculation: The beetles were inocu- dominant canopy species were Q. crispula, Q. ser- lated from 4 to 15 July 2003 (Table 1). The morn- rata, Castanea crenata Sieb. et Zucc., Magnolia ing of the inoculation day, we selected unwounded obovata Thunb., and Acer rufinerve Sieb. et Zucc. adult male P. quercivorus that had been held in The understory vegetation was composed of Ly- plastic containers for up to 3 d after collection. In onia neziki Nakai et Hara, Ilex pedunculosa Miq., the laboratory, each male was placed inside a 2 ml and Clethra barbinervis Sieb. et Zucc. An infesta- pipette tip, the point of which had been cut off tion of P. quercivorus was speculated to have af- where it was about 1.5 mm in diameter, and the fected this area in 2001, and approximately 50 large trees of Q. crispula had died by 2002. Table1.Day on which the beetles were inoculated Nine Q. crispula (20–30 cm DBH [diameter at Male Female breast height]) that had been infested with P. quercivorus and had died in October of 2002 were Uniform inoculation 04-Jul-03 07-Jul-03 felled at a site near the study area, and the trunks 07-Jul-03 11-Jul-03 of the felled trees were cut into 50 cm-long billets. 15-Jul-03 All the billets were brought to an outdoor cage at Random inoculation 07-Jul-03 11-Jul-03 the Kyoto Prefecture Forestry Experimental Station 11-Jul-03 15-Jul-03 (in the town of Wachi), and both ends of each billet Death of Quercus by Inoculation of the Beetles 125

wood. All treated trees were monitored for symptoms of disease (judged by the appearance of the leaves) almost every week after inoculation. When some of the leaves had begun to wither, but were not discolored completely, the inoculated tree was considered to be “wilted,” and the day on which the majority of leaves was completely discolored was considered to be the day of “death.” In late November, the trees were felled and the inoculated sections of the logs were cut into seven 3 cm-thick disks. We measured the areas of normal and discolored sapwood in the cross sections of Fig. 1. Uniform inoculation (compulsory inoculation of two disks taken from heights of 50 and 70 cm (the male adults via a pipette tips). The inoculation points: five top and bottom disks) and calculated the rate of rows at 3 cm intervals vertically with holes spaced at 3 cm intervals around the circumference. Two to three days after inoc- discoloration of the sapwood (the area of discol- ulating males in July, a female was introduced into each suc- ored sapwood/the area of normal and discolored cessful gallery made by a previously inoculated male. In late sapwood). In addition, the five central disks inocu- November, the rate of discoloration of the sapwood and the lated with P. quercivorus were split carefully with a density of galleries containing the next generation were calcu- hatchet, and the number of galleries that had suc- lated. cessfully produced offspring (N) was determined. The volume of wood from the height of 50 to base had been plugged with tissue paper. Each 70 cm (V) in which P. quercivorus were inoculated, pipette tip, containing one male, was put into a was computed from the diameter of the center disk, low-temperature box and carried to the study site and the density of the galleries containing the next after handling. generation (N/V) was calculated. The inoculation points were arranged in five Isolation of microorganisms. The two disks rows (3 cm apart vertically) at 3 cm intervals used to measure the area of discolored sapwood around the circumference (1 hole/33 cm, 50–60 were split, and eight small blocks (each 333 holes/tree). Holes 5 mm deep were drilled using a 3 mm) were cut from the discolored sapwood. The mm diameter bit just before inoculation (Fig. 1), surfaces of these blocks were sterilized for 5 min and each pipette tip carried to the study site was with a 2% sodium hypochlorite solution and rinsed thrust into a drill hole. After 2–3 d, one adult fe- three times in sterile distilled water. The surface- male was introduced via a pipette tip into each suc- sterilized blocks were placed on potato-dextrose cessful gallery made by a previously inoculated agar (PDA) and cultured at 22°C, and the rate of male (15–25 males/tree), and then the exposed isolation of R. quercivora (the number of isolated bark was covered with polyester cloth. blocks/8) was calculated. b. Random inoculation: The areas of another five trees where bark had been exposed were loosely RESULTS covered with polyester netting, and 200 adult male P. quercivorus were placed inside a net on each tree Four of the five trees that were uniformly inocu- from 7 to 11 July. After 2–3 d, one adult female lated died, as did all five trees that were randomly was inoculated via a pipette tip into each success- inoculated (Table 2). Seven of the nine dead trees ful gallery excavated by a previously inoculated showed the same wilting symptoms that are evident male (5–19 males/tree). in the current mass mortality of oak trees, i.e., they c. Control: As a control, the exposed bark of the withered completely within 1–2 wk after first de- remaining five trees was covered with polyester veloping symptoms. The two other dead trees (No. cloth after drilling holes in the same manner as for 1 and 3 from the random inoculation), which had uniform inoculation on 4 July, but no adult beetles no galleries that successfully produced offspring, were applied. had withered leaves that were light in color, al- Monitoring symptoms and surveying the sap- though the majority of leaves had discolored. In 126 H. KINUURA and M. KOBAYASHI

Table2. Monitoring for symptoms of disease

Inoculation DBH Gallery 15-Aug 25-Aug 5-Sep 8-Sep 12-Sep 16-Sep 29-Sep 7-Oct methods (cm) number

Uniform No. 1 9.2 11 H H H W DDDD Uniform No. 2 9.2 14 H W DDDDDD Uniform No. 3 9.2 5 HHHHWDDD Uniform No. 4 8.5 2 H H H W DDDD Uniform No. 5 10.0 7 HHHHHHWW

Random No. 1 7.8 0 H H H W W D D D Random No. 2 9.2 9 H H W DDDDD Random No. 3 9.5 0 HHHHHHDD Random No. 4 9.5 14 H H W DDDDD Random No. 5 10.2 6 HHHHHHDD

Control No. 1 8.2 0 HHHHHHHH Control No. 2 8.4 0 HHHHHHHH Control No. 3 8.6 0 HHHHHHHH Control No. 4 9.5 0 HHHHHHHH Control No. 5 9.9 0 HHHHHHHH

Gallery number: The number of galleries that contained offspring. H: Healthy, No leaves were changed. W: Wilted, Some leaves were discolored. D: Dead, All leaves were completely discolored.

Fig. 3. The relationship between the number of days from Fig. 2. The relationship between the number of days from inoculation to death and the rate of isolation of Raffaelea inoculation to death and the density of galleries containing off- quercivora. spring. contrast, the five control trees all appeared healthy. higher isolation rate of R. quercivora (Fig. 3; There was no evidence of attacks by P. r0.81, p0.01, logistic regression), and the quercivorus, except in the artificially inoculated density of beetle galleries that contained offspring parts of trees, and there were no attacks on control was positively related to the rate of discoloration of trees. Reproduction by P. quercivorus was con- the sapwood (Fig. 4; r0.66, 0.01p 0.05, logis- firmed in seven inoculated trees (Table 2). Trees tic regression) and the isolation rate of R. quer- that died faster had a greater density of beetle gal- civora (Fig. 5; r0.76, 0.01p0.05, logistic re- leries that contained offspring (Fig. 2; r0.70, gression). The four inoculated trees that showed 0.01p0.05). 100% discoloration withered earlier, within 65 d The pathogenic fungus R. quercivora was iso- after the inoculation. lated from all inoculated trees, but was not isolated from control trees. Trees that died faster had a Death of Quercus by Inoculation of the Beetles 127

Zealand remained uncertain. Quercus trees such as cork oaks in southern Europe wither after attacks by Platypus cylindrus (Fabricius) and it has been suggested that Raffaelea montetyi M. Morelet isolated from the mycangia of P. cylindrus is associated with the death of these trees (Cassier et al., 1996). However, no ambrosia beetles have been shown conclusively to regularly transmit pathogenic fungi, although it has been suggested or in- ferred. Here, we demonstrated that artificial inoculation Fig. 4. The relationship between the density of galleries with adult P. quercivorus leads to wilting death of containing offspring and the rate of isolation of Raffaelea oak trees. It is now clear that P. quercivorus trans- quercivora. fer the phytopathogenic fungus R. quercivora, which has resulted in the mass mortality of oak trees across Japan. This is the first report that a plant disease can be induced experimentally in healthy trees through attacks by an ambrosia beetle. ACKNOWLEDGEMENTS We thank Ms. A. Nozaki and other staff of the Kyoto Pre- fecture Forestry Experimental Station for help with the field- work. Thanks are also extended to Mr. K. Shuhara for his ad- vice on the selection of an appropriate study site. Fig. 5. The relationship between the density of galleries REFERENCES containing offspring and the rate of sapwood discoloration. Browne, F. G. (1961) The biology of Malayan Scolitidae and DISCUSSION Platypodidae. Malayan For. Records 22: 1–255. Cassier, P., J. Lévieux, M. Morelet and D. Rougon (1996) Many species of bark beetles are vectors of The mycangia of Platypus cylindrus Fab. and P. o xyurus plant-pathogenic fungi, including Scolytus spp., Dufour (Coleoptera: Platypodidae). Structure and associated fungi. J. Insect Physiol. 42: 171–179. which are responsible for Dutch elm disease (Web- Faulds, W. (1977) A pathogenic fungus associated with ber and Gibbs, 1989). However, ambrosia beetles Platypus attack on New Zealand Nothofagus species. N. like P. quercivorus usually prefer wilting or dying Z. J. For. Sci. 7: 384–396. trees, and it is unusual for them to attack healthy Ito, S. (2002) Recent problems in death and decline of the living trees (Kajimura, 2002). Therefore, it is very fagaceous tree species. Shinrin Kagaku 35: 4–9 (in Japanese). rare for a platypodid beetle to be a vector of a Ito, S., T. Kubono, N. Sahashi and T. Yamada (1998) Associ- pathogen (Browne, 1961). ated fungi with the mass mortality of oak trees. J. Jpn. Three species of Platypodidae in New Zealand For. Soc. 80: 170–175 (in Japanese with English sum- are suspected of being vectors of Sporothrix spp., mary). the pathogenic fungi that kill Nothofagus spp. Ito, S. and T. Yamada (1998) Distribution and spread of the (Faulds, 1977). However, Platypus subgranosus mass mortality of oak trees. J. Jpn. For. Soc. 80: 229–232 (in Japanese). (Schedl), which infests living Nothofagus cunning- Kajimura, H. (2002) Colonization patterns and reproductive hamii (Hook.) Oerst. in Australia, was shown not traits of Scolytidae and Platypodidae with special refer- to be an important vector of the plant-pathogenic ence to ambrosia beetles. Jpn. J. Ecol. 52: 81–88 (in fungus Chalara australis Kile et Walker, and bee- Japanese). tles attacked trees that were already infected by C. Kile, G. A. and M. F. Hall (1988) Assessment of Platypus subgranosus as a vector of Chalara australis, causal australis (Kile and Walker, 1987). Therefore, Kile agent of a vascular disease of Nothofagus cunninghamii. and Hall (1988) noted that the role of P. subgra- N. Z. J. For. Sci. 18: 166–186. nosus in the death of N. cunninghamii in New Kile, G. A. and J. Walker (1987) Chalara australis sp. nov. 128 H. KINUURA and M. KOBAYASHI

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