THE PRIMARY AND AUXILIARY AMBROSIA FUNGI ISOLATED FROM THE AMBROSIA BEETLES, Title SCOLYTOPLATYPUS SHOGUN BLANDFORD (COLEOPTERA SCOLYTIDAE) AND CROSSOTARSUS NIPONICUS BLANDFORD(COLEOPTERA PLATYPODIDAE) Author(s) NAKASHIMA, Toshio; GOTO, Chie; IIZUKA, Toshihiko Citation Journal of the Faculty of Agriculture, Hokkaido University, 63(2), 185-208 Issue Date 1987-03 Doc URL http://hdl.handle.net/2115/13057 Type bulletin (article) File Information 63(2)_p185-208.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP THE PRIMARY AND AUXILIARY AMBROSIA FUNGI ISOLATED FROM THE AMBROSIA BEETLES, SCOLYTOPLA TYPUS SlfOaUN BLANDFORD (COLEOPTERA: SCOLYTIDAE) AND CROSSOTARSUS N1PON1CUS BLANDFORD (COLEOPTERA: PLATYPODIDAE) Toshio NAKASHIMA* , Chie GOTO** and Toshihiko IIZUKA (Department of Agricultural Biology, Faculty of Agriculture, Hokkaido University, Sapporo 060 JAPAN) Received October 21, 1986 Abstract An ambrosia fungus and several kinds of yeasts associated with the ambrosia beetles Scolytoplatypus shogun Blandford and Crossotarsus niponicus Blandford were isolated and identified. A specific ambrosia fungus, Am brosiella sp., was isolated from both the mycetangia and the galleries of S. shogun. Pichia spp. were also isolated from the galleries of the beetles. It is considered that the fungus, Ambrosiella sp., is the primary ambrosia fungus and the Pichia spp. are the auxiliary ambrosia fungi of S. shogun, respectively. Two kinds of yeasts, Endomycopsis platypodis and Torulopsis norvegica. were commonly isolated from both mycetangia and galleries of C. niponicus. These fungi seem to be the primary ambrosia fungi of this beetle. The morphological and physiological characteristics of these isolates were described. The range of the optimum temperature of Ambrosiella sp. was extremely narrow at approximately 23°C. For E. platypodis and T. norvegica, the range of the optimum temperature was relatively wide from 20°C to 30°C. Introduction It is well known that ambrosia beetles carry viable fungus spores within small organs called mycetangia in their bodies. When an ambrosia beetle [J. Fac. Agr. Hokkaido Univ., Vol. 63, Pt. 2. 1987] * Emeritus Professor of Hokkaido University. ** Hokkaido National Agricultural Experiment Station, Memuro-Cho, 082 JAPAN. 186 T. NAKASHIMA, C. GOTO AND T. IIZUKA tunnels into wood, these viable spores are dislodged from the mycetangia, and a mass of velvety fungus soon lines the interior of the tunnel. Until the present time, many species of fungi, more than 30 genera, have been reported as the fungi associated with ambrosia beetles. Only few species of these fungi, however, have been demonstrated as the true mutualistic symbiont or ambrosia fungi of these beetles. In this paper, we have reported that one species of ambrosia fungus and several kinds of species of yeasts were isolated from mycetangia and galleries of Scolytoplatypus shogun, and several kinds of yeasts were isolated from the mycetangia and galleries of Crossotarsus niponicus. The morphological and physiological characteristics of each species of these isolates have been described and identified. Materials and Methods Collection of beech logs infested with the beetles. The ambrosia beetles used in the present study were Scolytoplatypus shogun as Scolytidae and Crossotarsus niponicus as Platypodidae. Beech logs (Fagw crenata Blume) infested with the beetles were collected at the Hiyama Forest Experiment Station of Hokkaido University, Kamino kuni, Hiyama in September 1980 and July 1981, and immediately carried to the campus of the Faculty of Agriculture, Hokkaido University in Sapporo. The logs collected in the fall were overwintered under snow on the campus and were used in the next spring. Isolation of microorganisms from galleries of the beetles. The surface of the pinholed beech logs were washed by ethyl alcohol and burned. After that, the logs were cut into small pieces of about 2 cm3 , and the adults and larvae of the beetles were aseptically removed from the TABLE l. Composition of media. Substance YM broth (g) eM broth (g) Glucose 10 20 Peptone 5 10 Yeast extract 3 5 Malt extract 3 MgS0-7H O 2 4 2 K2HP04 5 Distilled water 1000 ml 1000 ml AMBROSIA FUNGI ISOLATED FROM AMBROSIA BEETLES 187 galleries. The inner surface of each gallery lined with velvety fungi was washed with TM and CM broth for culture. Components of both broth are shown on Table 1. Collection of the adults. In the overwintering period, the adults of S. shogun were collected from pinholed beech logs. In the flying period, the new adults of each species were collected within IS hours after appearance. Culture conditions for isvlates. i). The galleries were washed by YM and CM broth. After that, each broth was basically incubated by shaking culture at 25°C for five days, and then the cultures were incubated on YM and CM agar plates. ii). Velvety fungus from the surface of the galleries was directly inocu lated on both agar plates. iii). The surface of each gallery was washed by sterile distilled water and drop of that water was incubated on both agar plates. Isolation of microorganisms from the mYcetangia of beetles. The mycetangium of S. shogun is a small tissue located under the surface of the center of the pronotum of female adults. The shape of this tissue is like a jellyfish (Fig. 1)8,9). This tissue is easily separable from other tissues under microscope. The mycetangium of C. niponiclls is a bag around a sphere-shaped tissue located at the back of the preoral cavity of female adults (Fig. 2)6,7,9). This mycetangium is cleanly inseparabled, therefore, in the case of C. niponicu~, the mycetangium was cut out with another tissues in the head attached. The mycetangia of each species were aseptically placed in YM and CM Fig.!. The mycetangium of S. shogun. 188 T. NAKASHIMA, C. GOTO AND T. IIZUKA Fig. 2. The mycetangium of C. niponiclls. broth on slide glass and then were incubated on agar plates. Small amounts of the broth on slide glass were microscopically observed after incubation. Isolation of microorganisms from the homogenates of larvae. The larvae of each species were collected from pinholed beech logs onto cheese cloth and washed twice with distilled water. Each larva was homo genized gently in 1 ml of YM broth with sterile teflon homogenizer. The homogenates were spread over YM agar plates and incubated at 2.5°C up to form colonies. Isolation of microorganisms from the digestive tracts of adult beetles in flight season. The digestive tracts of new adults were dissected and incubated on YM agar plates. Identification of the isolates. Isolated yeasts were basically identified according to LODDERo), and ISO, lated ambrosia fungi were identified according to BATRA2). Scanning electron microscopy. Scanning electron microscopy of the ambrosia fungi and the wall of the galleries of S. shogun was obtained by a 2% OS04 vapor fixation fol· lowed by coating with carbon and gold. Samples were observed by the scanning electron microscope Type JSM-SI. Results 1. Fungi associated with Scolytoplatypus shogun. Isolated fungi from mycetangia of adult beetles, homogenates of larvae, TABLE 2. The fungi isolated from S. shogun. !l> ~ Adult female Larvae Walls of galleries to I o::<l Condition of isolation Nsetgwa.yaalmldegur ~ymt s Flying period I Bpreeeridoidn g Homo- Owvienrt-ering New egg Breeding Egamllpetryy U>l Mycetangia 1D igestive 1 Body genates period cradles period ejmusetr gaefntecre c'T:j tracts surface z Season May June June 1J ~l.-AUg·1 July 1IM-~ -.5-M-a1y 1 June July Aug. HC l H Number of tested 20 27 10 10 4 2 3 1 o(fJ 1 1 t"" Number of strains isolated 8 23 1 5 7 23 10 9 2 ~ 1 1 1 1 tlj t:I Ambrosiella sp. ffiI ffiIffiI - - - - lIIII ffiI - 'Tj Candida tenuis - - - - - lIIII - - - o::<l Pichia sp. 1. - - - + + ffiI - * - ~ Pichia sp. 2. - - - * -lit * - * + !l> ~ Pichia sp. 3. - - + - - - - - - :ot:o<l Endomycopsis sp. - - - - - + - - - >(fJ I unknown sp. 1. + - - - - - - - - to unknown sp. 2. + - - - - - - - - tlj white fungus * * -- + - + - + - ~ t"" - tlj Ul Numbers of (+) indicate numbers of strains isolated. f-' (Xl <.0 190 T. NAKASHIMA, C. GOTO AND T. IIZUKA TABLE 3. Morphological characteristics of Ambrosiella sp. isolated from the new adults of S. shogun. Ambrosiella sp. Colony characteristics: Young colonies on YM agar at 25°C are white, 3 days old colonies are white to cream-colored, 1 cm in diameter, the center of the colony becomes elevated 1.5 mm high 2.0 mm in diameter; marginal mycelium hyaline to sub hyaline, detached, becoming dendroid; superficially white aerial hyphae are formed. 7 days old colonies are greyish green, 4 cm in diameter, with a fruity odor, the center of the colony become elevated, reddish brown pigment oozing as small droplets on masses of aerial hyphae, aerial hyphae are white to dark grey, undersurface dark green. 14 days old colonies are dark grey, 5.5 cm in diameter, with a stimulative odor, a reddish brown zone of diffusion present, simmilar pigment also oozing in the form of small droplets on the mycelium; mycelium white to dark grey, superficially wooly aerial hyphae, at places sporo dochia appear. Microscopic characteristics: Hyphae hyaline to reddish brown, repeatedly branched, becoming dendroid sometimes irregular form. Conidia brastosporic, globose to subglobose, subhyaline to reddish brown, thick-walled, contain many granules, brone singly or in monilioid chains, smootb-walled, measure 11-28 fl in diameter. Optimum growth temperature: 23°C. TABLE 4. Morphological and physiological characteristics of Candida tenuis isolated from the gallery of S. shogun. Candida tenuis Diddens et Lodder Growth in YM broth: After 3 days at 25°C, the cells are oval, long-oval to elongate, 1.2-7.0 fl X 1.2-18.0 fl, single, in pairs or in short chains, ring and sediment. Streak culture on YM agar: After 1 month at 25°C, cream-colored, punctate, dull, mucoid, raised, border filamentations. Dalmau plate culture on corn-meal agar: Pseudomycelium well developed. No true mycelium. Blastospore present. Fermentation: Glucose + Sucrose Lactose Galactose -1:- Maltose Raffinose Assimilation of carbon compounds: Glucose + Sucrose + Melibiose Galactose + Maltose + Raffinose L-Sorbose + Trehalose + Inositol Assimilation of KN03: Negative. AMBROSIA FUNGI ISOLATED FROM AMBROSIA BEETLES 191 TABLE 5. Morphological and physiological characteristics of Pichia sp. 1. isolated from the homogenates of the larvae of S. shogun. Pichia sp. 1. Growth in YM broth: After 3 days at 25°C, the cells are globose, 1.2-5.0 fl, single, in pairs or in clusters. After 1 month no pellicle but ring and mucoid sediment. Streak culture on YM agar: After 1 month at 25°C, cream-colored, smooth, shiny, mucoid, cross-section convex, bordes entire. Dalmau plate culture on corn-meal agar: No pseudo mycelium formation. Sporulation: Spores formed easily on YM agar. Asci are globose, contain 2 to 4, usually 4 ascospores. Ascospores are hat-shaped, spheroidal or hemicycle, which have short brim. Fermentation: Absent. Assimilation of carbon compounds: Glucose + Maltose Lactose Galactose Cellobiose + Melibiose Sucrose Trehalose + L-Arabinose + (weak) Assimilation of KN03: Negative. TABLE 6. Morphological and physiological characteristics of Pichia sp. 2. isolated from the homogenates of the larvae of S. shogun. Pichia sp. 2. Growth in YM broth: After 3 days at 25°C, the budding yeast cells are spherical, oval and cylindrical, 1.2-S.0x1.2-12.5 fl, and occur singly or in pairs. A sediment and a ring are present. After one month at 25°C, a sediment and a ring are present. Growth in YM agar: After 3 days at 25°C, the budding yeast cells are spherical to oval, cylindrical, 1.5-5.0x1.5-10.0 fl, and occur singly or in branched chains. After one month at room temperature the streak culture is cream-colored, dull, butyrous, smooth or slightly wrinkled, with a undulate partly filamentous margin. Dalmau plate culture on corn-meal agar: A primitive pseudo mycelium is abundantly formed. It consists of oval chain cells and of a tree-like appearance. Formation of ascospore: Asci are oval to long-oval. The spores are hat shaped, usually four are formed per ascus. They are easily liberated from the ascus. Sporulation is good in YM broth, on YM agar and cornmeal agar. Fermentation: Glucose + Sucrose Lactose Galactose Maltose Raffinose Trehalose Assimilation of carbon compounds: Glucose + Maltose + Melibiose Rhamnose + Galactose + Cellobiose Raffinose Ethanol + L-Sorbose + Trehalose Xylose +(weak) Mannitol + Sucrose +(weak) Lactose L-Arabinose Inositol Assimilation of potassium nitrate: Negative. 192 T. NAKASHIMA, C. GOTO AND T. IIZUKA TABLE 7. Morphological and physiological characteristics of Pichia sp. 3 isolated from the digestive tracts of the adult female of S. shogun. Pichia sp. 3. Growth in YM broth: After 3 days at 25°C, the cells are oval, cylindrical to elongate, 1.5-5.0 flx2.0-16.0 fl, single, in pairs or in short chains; heavy ring, islets and sediment are present. After 1 month at 25°C, ring, islets and flaky sediment are present. Streak culture on YM agar: After 1 month at 25°C, cream-colored, wrinkled, dull, butyrous, raised, border filamentous. Dalmau plate culture on corn-meal agar: Pseudo mycelium well developed and blastospore present. Formation of ascospores: Ascospores are hat-shaped. Formation of ascos pores is poor on YM agar. Fermentation: + + Glucose Maltose Raffinose + + Galactose Trehalose + Sucrose Lactose Assimilation of carbon compounds: Glucose + Cellobiose + Raffinose + + + Galactose Trehalose Xylose Sucrose + Lactose Inositol + Maltose Melibiose Assimilation of KN03: Negative. TABLE 8. Morphological and physiological characteristics of Endomycopsis sp. isolated from the gallery of S. shogun. Endomycopsis sp. Growth in YM broth: After 3 days at 25°C, the cells are ovar, cylindrical, elongate, 1.2-6.0 fl X 2.5-28.0 fl, single or in pairs; no ring and no pellice but flaky sediment. After 1 month indistinct ring, incomplete thin pellicle and floc culent sediment. Budding is on a broad base. Streak culture on YM agar: After 1 month at 25°C, beige colored with reddish tinge, verrucose, dull, leathery, raised, border filamentous. Dalmau plate culture on YM agar: Psudomycelium and true mycelium well developed. Formation of ascospore: The asci are situated terminally or laterally on the hyphae, long-oval or elongate, contain 4 ascospores. Ascospores are hat shaped. Fermentation: Absent. AMBROSIA FUNGI ISOLATED FROM AMBROSIA BEETLES 193 and their galleries of S. shogun are shown in Table 2. Morphological and physiological characteristics of these isolates were investigated and identified as shown from Table 3 to Table S. Morphological characteristics are also indicated from Fig. 3 to Fig. 10. Ambrosiella sp., which was classified by BATRA2) as the specific ambrosia fungus, was isolated from 19 individuals out of 27 beetles tested, and they were isolated purely without another microorganisms except two individuais. The scanning electron microscopy of Ambrosiella sp. from mycetangia and galleries is shown in Fig. 11 and Fig. 12. Tn addition to the isolation of Ambrosiella sp., numerous strains of (d) ( a) (e) 10p 10p Fig. 3. Ambrosiella sp. (a) After 2 days YM slant culture. (b) After 5 days YM slant culture. (c) After 3 days Corn meal slide culture. (d) Chains of ambrosia cells and conidia around egg of Scolytoplatyj>us shogun. (e) Chains of cells from mycetangia of S. shogun.