植物研究雑誌 J. Jpn. Bot. 83: 77–87 (2008)

In vitro Symbiosis between Gastrodia elata Blume (Orchidaceae) and Armillaria Kummer (Tricholomataceae) Species Isolated from the Orchid Tuber

Gen KIKUCHIa,＊, Masami HIGUCHIa, Hiroaki YOSHIMURAa, Takashi MOROTAa, Akira SUZUKIb

aBotanical Raw Materials Research Department, Botanical Raw Materials Division, Tsumura & Co., 3586, Yoshiwara, Ami-machi, Ibaraki, 300-1192 JAPAN; ＊E-mail: [email protected] bFaculty of Education, Chiba University, Chiba, 263-8522 JAPAN (Recieved on August 20, 2007)

An Armillaria species isolated from Gastrodia elata was identified as A. gallica based on morphological characteristics of basidiomata and mating test. Symbiosis be- tween G. elata and A. gallica was confirmed by using two-member cultures. Hyphal coils of the fungal symbiont were formed in the cortical cells of tubers and isolated from their cortical cells. Length and weight of fungus-infected tubers were significantly higher than those of non fungus-infected tubers and control (P < 0.05). These indicated that G. elata has a mycorrhizal symbiotic relation to A. gallica.

Key words: Armillaria, Gastrodia elata, mycorrhiza, symbiosis, two-member culture.

Gastrodia elata Blume is an achloro- report has been done to clarify the symbiosis phyllous orchid that is unable to exist as an experimentally by using in vitro two- autophyte (Fig.1). Gastrodia elata therefore member culture. makes symbiotic mycorrhiza with Armillaria Terashita (1985) reported a two-member species and lives together (Kusano 1911). culture between Galeola septentrionalis The tuber of G. elata is used as a natural Rchb. f. and Armillaria. Tashima et al. medicine for headache and vertigo as (1978) recognized the symbiosis between “Tenma” in Kampo medicine (Hatakoshi Gastrodia verrucosa Blume and an unidenti- 1947, Japanese Pharmacopoeia 2006) and as fied fungus in vitro.Inorder to prove the “Tian-ma” in Chinese medicine (Pharma- symbiosis between achlorophyllous orchid copoeia of the People’s Republic of China and fungus, it is necessary to carry out two- 2005). Though it has been produced by member culture. cultivation in China, there still remain many The objects of this study are: to recognize ambiguous issues about the symbiosis. the symbiosis between G. elata and The association between G. elata and Armillaria by using two-member culture in Armillaria has been reported by Kusano vitro;toinvestigate the vegetative reproduc- (1911) and Hamada (1940). Cha and Igarashi tion process of G. elata, and to identify the (1995) reported five Armillaria species ob- symbiont of G. elata in Honshu, Japan. tained from G. elata tubers. However, these studies were conducted based on materials from a limited area in Hokkaido and no

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dug with soil and were washed carefully. There were two types of young-stage tubers. One was adhered by rhizomorphs of Armillaria species on the surface and the other was not. The former type collected in 2000 was used for isolation of fungal symbiont and the latter collected in 2001 was used for the association experiment (Table 1). For the subsequent experiments, all the tubers were washed twenty times with steril- ized water to avoid contamination.

Isolation of the fungal symbiont Several tubers adhered by rhizomorphs were selected for fungal isolation. After washing, the tubers were cut into serial 3 mm segments with a sterilized scalpel. The fungal symbiont was isolated by picking up hyphal coils from the cortex cells of the tuber using a sterilized platinum loop under a dissecting microscope and transferring these structures to Petri dishes containing po- tato-dextrose-agar medium (PDA, Difco). The Petri dishes were incubated at 25ºC in the dark.

Fig. 1. Gastrodia elata in the field. The photograph Basidioma formation of symbiont was taken at Kessoku-cho, Ushiku City, Ibaraki One hundred and twenty milliliters of the Pref., Japan, on 4 June 1995. sawdust medium (beech sawdust: rice bran 3:1;60
moisture content) was put into a Materials and Methods 300 mL plastic cultivation bottle. An inocu- Collection of Gastrodia elata tuber lation hole of 15 mm in diam. was made in Tubers of G. elata were collected in 2000 the center of the medium. Then the bottles and 2001 from Quercus acutissima forests in were autoclaved at 121ºC for 15 min. Ryugasaki City, Ibaraki Prefecture, Japan. Ground carrot (5 g) autoclaved in the same To collect young tubers, whole plants were way, was put into the bottle of the inocula-

Table 1. Mycorrhiza formation and rhizomorphs adhetion to Gastrodia tubers

Tubers in the field Tubers in vitro Rhizomorphs observed mycorrhizal observed mycorrhizal Adhesion 19 9 26 19 Non adhesion 104 0 0 0 Total 123 9 26 19 April 2008 Journal of Japanese Botany Vol. 83 No. 2 79

Table 2. Haploid testers of Armillaria species and their origins in Japan

Biological species Host tree Locality A. gallica Abies veitchii Mt. Simagareyama, Nagano Pref. A. veitchii Mt. Simagareyama, Nagano Pref. A. cepistipes Cryptomeria japonica Mogami Town, Yamagata Pref. C. japonica Mogami Town, Yamagata Pref. A. ostoyae Chamaecyparis obtusa Siwa Town, Iwate Pref. C. obtusa Iwate Town, Iwate Pref. A. tabescens Quercus acutissima Ami Town, Ibaraki Pref. Q. acutissima Ami Town, Ibaraki Pref. tion hole. Two grams of the fungal autoclaved at 121ºC for 15 min. Inoculation inoculums grown on PDA medium were put plugs of symbiont 1 cm in diam. were inocu- on the carrot. The cultivation bottles were lated on the sawdust. The culture was incu- kept at 25.0
0.5ºC for 40 days in the dark. bated at 25.0
0.5ºC for 6 months until fungal For fruiting induction, the bottles were sub- rhizomorphs were formed actively. Eight jected to 16.0
1.0ºC under intermittent illu- cultures were made in this way. mination with 350 lx alternated with the dark Three or four tubers without rhizomorph every 12 hours (Togashi and Takizawa adhesion were put into the incubating flask. 1992). Four replicates were made. The tubers were covered with the sawdust and incubated at 15
0.5ºC for 3 months in Mating test the dark. As the control, eight tubers were in- The haploid isolates of fungal symbiont cubated in two flasks in which no fungal were each isolated from a single spore of symbiont was inoculated. After the incuba- basidioma. The single spore isolates of tion, the tubers were examined in length, symbiont were mated with haploid tester weight, adhesion of rhizomorphs, and strains of Armillaria species isolated from mycorrhizal formation. For confirmation of the basidioma collected in Japan (Table 2). the mycorrhizal peloton, tubers were sec- Mating experiments were performed by plac- tioned and observed under a dissecting mi- ing inocula 1 mm apart on a 1.25
malt ex- croscope and scanning electron microscope tract agar and incubated at 25
0.5ºC for 4 (SEM). Mycorrhizal peloton was isolated weeks in the dark. The single spore isolates from the tuber using a dissecting microscope of an Armillaria symbiont were mated with and cultured at 25.0
0.5ºC for 2 weeks on haploid tester strains of several Armillaria PDA. species. The tester strains were first paired in all combinations in order to assay the pattern Results of the appearance of compatible or incom- Gastrodia elata in the field patible pairings. Results of the mating tests Host trees of fungal symbiont were not de- were judged according to Korhonen (1978). termined by tracing rhizomorphs because The pairings were conducted in triplicates. many rhizomorphs were distributed over the forest at the depth of 10–15 cm. Rhizo- Association experiment in the sawdust morphs developed well around flowering medium tubers. Many tubers of G. elata grew around Fifty grams of the sawdust medium was a decayed mother tuber located just under placed in a 300 mL Erlenmeyer flask and the flowering tuber. The flowering tuber and 80 植物研究雑誌 第83巻第2号平成20年4月

(Fig. 4). In total one hundred and twenty- three tubers were collected in the field. Among them were nineteen rhizomorph- attached tubers, and nine mycorrhiza-formed ones (Table 1). Mycorrhizal formation was observed only in rhizomorph-attached tubers.

Isolation of the fungal symbiont After 3 weeks cultivation all isolates from the hyphal coils grew well and produced dark brownish crustose colonies on PDA me- dium. They were confirmed to be Armillaria sp. based on colony morphology, mycelial growth and rhizomorph formation on PDA plates. One of these cultures was used for subsequent examinations. Fig. 2. An illustration of tuber growth pattern of Gastrodia elata. Daughter tubers grow around a Identification of Armillaria species decayed mother tuber located under the flowering Following dark cultivation a crustose tuber. AS. Aerial stem. DT. Daughter tuber. FT. mycelium was formed on the surface of Flowering tuber. MT. Mother tuber. R. the sawdust in the 300 ml cultivation plastic Rhizomorph. S. Soil. I. Inflorescence. bottle. Basidiomata were formed on the mycelium for a further 3 weeks incubation other tubers were budded from the mother under the light. Scales on the pileus were tuber that was attached and penetrated by dark gray. Base of the stipe was swollen. rhizomorphs (Figs. 2, 3). Annulus was thin, arachnoid and evanescent Some daughter tubers were attached by (Fig. 5). These characteristics corresponded rhizomorphs extended from the mother tuber to those of Armillaria gallica Marxmuller &

Fig. 3. Many daughter tubers (arrows) formed around the mother tuber. These tubers are present under the flowering tuber of Gastrodia elata. Bar = 2 cm. April 2008 Journal of Japanese Botany Vol. 83 No. 2 81

Fig. 4. Daughter tubers attached and penetrated by rhizomorphs extended from the mother tuber. Bar=4mm.

Fig. 5. Basidiomata of Armillaria gallica. The basidioma was formed after three weeks incubation under the light.

Romagnesi. The symbiotic Armillaria isolated from G. Results of the mating tests are shown in elata tuber was compatible with testers of A. Table 3. The fluffy mycelium of fungal sym- gallica. biont developed into a crustose mycelium. 82 植物研究雑誌 第83巻第2号平成20年4月

Table 3. Mating test of haploid isolates between mycorrhizal Armillaria testers

Tester species and their sexual type Mycorrhizal symbiont A. gallica A. cepistipes A. ostoyae A. tabescens (Sexual type) 12 12 12 12 1 + + – – – – – – 2 + + – – – – – – 3 + ? – – – – – – 4 + ? – – – – – –

+: Compatible pairing; –: Incompatible pairing; ?: Uncertain pairing.

Fig. 6. Gastrodia elata tuber cultured with Armillaria gallica on the sawdust medium for three months. Budding tubers were observed on the inocu- lated tuber (it) adhered by the the rhizomorphs. Bar=3cm.

Association experiment in the sawdust duced new tubers actively. On the other medium hand, multiplication of non-mycorrhizal In Armillaria-inoculated bottles, rhizo- tubers was very limited (Figs. 10, 11). In the morphs were formed on the sawdust cover- control, no rhizomorphs adhered to tubers ing the tuber by 3 months cultivation. All nor was mycorrhiza formation observed. the tubers were adhered by the rhizomorphs Though they formed some buds, the number to the surface (Table 1) and formed new was smaller than those of the inoculated buds (Fig. 6). Of these, 19 tubers showed tubers (Fig. 7). mycorrhiza (Figs. 8, 9). Planted tubers did The mean length of inoculated tubers was not grow themselves but produced new tu- 7.5 times longer and mass was 4 times heav- bers. Therefore, the length of tubers was ier than those of the control (P < 0.05 Table shown in the total length of original and bud- 4). ding new tubers. Mycorrhizal tubers pro- There were significant differences April 2008 Journal of Japanese Botany Vol. 83 No. 2 83

Fig. 7. Gastrodia elata tubers cultured without Armillaria on the sawdust medium for 3.5 months. A few stem- like organ (arrows) bud from the inoculated tuber (it). Bar = 2 cm.

Table 4. Size of Gastrodia elata tubers incubated with Armillaria gallica for three months

Fungal treatment Number of tested tuber Length (mm) Fresh mass (g) Inoculated 26 75.8＊
(3.6) 0.8＊
(0.05) Control 8 10.0
(1.8) 0.2
(0.04)

＊: Significant difference between inoculated tuber and control P < 0.05. Number in paren- theses show standard error

Fig. 8. Mycorrhiza formed on Gastrodia elata tuber. rz. Rhizomorphs. m. Mycorrhiza. p. Parenchyma. ep. Epidermis. Bar = 500 m. 84 植物研究雑誌 第83巻第2号平成20年4月

Fig. 9. Scanning electron micrograph of hyphal coils of an Armillaria symbiont in cortical cells of Gastrodia elata tuber observed 3 months after incubation in sawdust medium. cw: Cell wall. m: Mycelial coils. Bar=20
m.

between mycorrhizal and non-mycorrhizal tubers. Therefore adhesion of rhizomorphs to tubers, and between the mycorrhizal tubes tubers occurred prior to mycorrhizal forma- and control in length, though there was no tion both in the field and in vitro. These difference between non-mycorrhizal tubers tubers were attached and penetrated by and control (P < 0.05, Fig. 10). There were rhizomorphs. Mycorrhiza were observed as also significant differences in mass between hyphal coils within cortical cells of these mycorrhizal and non- mycorrhizal tubers and tubers, while these coils were not observed between the mycorrhizal tubers and control from other parts of tubers because they have (P < 0.05, Fig. 11). been digested in this area (Hamada 1958). The symbiotic fungi were isolated from There were significant differences be- cortical cells of the cultivated tuber again. tween mycorrhizal and non-mycorrhizal The culture was regarded as an Armillaria tubers, and between the mycorrhizal tubers species based on its colony morphology, and control in length and mass though there mycelial growth and rhizomorph formation. was no difference between non-mycorrhizal tubers and control. Mycorrhizal tubers pro- Discussion duced a lot of new tubers though non- There has been no report experimentally mycorrhizal tubers and control produced clarify the symbiosis between G. elata and few tubers. This suggests that the tuber of Armillaria using in vitro two-member cul- G. elata obtains nutrition by digesting ture. In this study the symbiotic relationship hyphal coils of A. gallica.Onthe other hand, between Gastrodia elata and Armillaria it is not clarified whether Armillaria obtains gallica was proved with the two-member an advantage from G. elata. The daughter culture. All of the mycorrhizal tubers were tubers were not adhered by the rhizomorphs observed only in rhizomorph- attached at first. Rhizomorphs from mychorrhizal April 2008 Journal of Japanese Botany Vol. 83 No. 2 85

Fig. 10. Length of Gastrodia tubers incubated with Armillaria gallica. ＊P<0.05 vs. non mycorrhizal tubers; +P < 0.05 vs. control.

Fig. 11. Weight of Gastrodia tubers incubated with Armillaria gallica. ＊P<0.05 vs. non mycorrhizal tubers; +P < 0.05 vs. control.

mother tuber extended to daughter tubers and plant pathogen (Thomas 1934). Mating tests adhered to them. Consequently daughter have been commonly used for the identifica- tubers were infected by symbiont. This tion of Armillaria species. Several species propagation mode was the same in the field have been delimited in Europe (Korhonen and in vitro. 1978, Guillaumin et al. 1993), North Armillaria is well known as an important America (Anderson and Ullrich 1979), 86 植物研究雑誌 第83巻第2号平成20年4月

Australia (Kile and Watling 1983), and The authors are grateful to Dr. M. Okada Africa (Mwangi et al. 1989). Japanese spe- (The Kochi Prefectural Makino Botanical cies of Armillaria have been examined by Garden) and Dr. S. Terabayashi (Yokohama Nagasawa et al. (1991), Suzuki et al. (1994), College of Pharmacy) for helpful discus- Cha et al. (1992, 1994, 1995) and Ota et al. sions. We thank for E. Hasega for providing (1998). At least 10 species are recognized in samples of biological species of Armillaria. Japan. Armillaria gallica was well-known as We also thank to Mr. S. Minoura, Mr. N. a saprobic species and produced abundant Igari, Mr. M. Tanaka and the members of rhizomorphs in soil (Roll-Hansen 1985). Botanical Raw Materials Division, Tsumura Suzuki (1994) reported that A. gallica is a & Co., for useful information and their sug- weak pathogen of conifer forests and lived to gestions. decay dead hardwoods and litter, on the other hand, A. ostoyae is a strong pathogen References and lived to decay healthy coniferous trees. Anderson J. B. and Ullrich R. C. 1979. Biological spe- Ota et al. (1998) showed A. gallica would be cies of Armillaria mellea in North America. a weak pathogen and is found most com- Mycologia 71: 402–414. monly in the field, while A. ostoyae and A. Cha J. Y. and Igarashi T. 1995. Armillaria species as- sociated with Gastrodia elata in Japan. Eur. J. For. mellea seem to be strong pathogens in Japan. Path. 25: 319–326. The association between Gastrodia elata Cha J. Y., Sung J. M. and Igarashi T. 1992. Biological and Armillaria species was reported by species and morphological characteristics of Kusano (1911) and Hamada (1940), how- Armillaria mellea complex in Hokkaido: ever, they did not identify the Armillaria at A. ostoyae and A. bulbosa. Res. Bull. Exp. For. Hokkaido Univ. 49: 185–194. specific level. Cha and Igarashi (1995) re- Cha J. Y., Sung J. M. and Igarashi T. 1994. Biological ported that the Armillaria species isolated species and morphological characteristics of from G. elata in Hokkaido were A. gallica, Armillaria mellea complex in Hokkaido: A. ostoyae (Romagnesi) Herink, A. jezoensis A. sinapina and two new species, A. jezoensis and Cha & Igarashi, A. sinapina Berube & A. singula. Mycoscience 35: 39–47. Dessureault, and A. singula Cha & Igarashi. Cha J. Y., Sung J. M. and Igarashi T. 1995. Armillaria mellea (Vahl: Fr.) Kummer s. s. from Hokkaido. J. Among them A. gallica was most common. Jpn. For. Soc. 77: 395–398. In this study the symbiont of G. elata in Guillaumin J. J., Mohammed C., Anselmi N., Honshu, Japan was A. gallica. This result Courtecuisse R., Gregory S. C., Holdenrieder O., agrees with the case observed in Hokkaido Intini M., Lung B., Marxmüller H., Morrison D., (Cha and Igarashi 1995). Rishbeth J., Termorshuizen A. J., Tirró A. and Van Dam B. 1993. Geographical distribution and ecol- Mohammed et al. (1994) suggested that ogy of the Armillaria species in western Europe. A. ostoyae was probably too aggressive for a Eur. J. For. Pathol. 23: 321–341. symbiotic association with orchids and that Hamada M. 1940. Physiologisch-morphologische A. gallica was the most common symbiont studien über Armillaria mellea (Vahl) Quél., mit species with G. elata. They thought that this besonderer Rücksicht auf die Oxalsäurebildung. species produced abundant rhizomorphs in Ein Nachtrag zur Mykorrhiza von Galeola septentrionalis Reichb. f. Jpn. J. Bot. 10: 387–463. the soil and was a weak pathogen. These Hamada M. 1958. Mykotrophische Symbiose. Trans. features probably have the advantage to form Mycol. Soc. Jpn. 7: 2–9. the symbiotic relation. It is thought that the Hatakoshi M. 1947. Illustrations of Medicinal Plants. symbiosis of G. elata and Armillaria species Tokyonougyousyoin Tokyo (in Japanese). is formed with the balance of continuously Kile G. A. and Watling R. 1983. Armillaria species from south-eastern Australia. Trans. Br. Mycol. formed mycorrhiza and mycorrhizae diges- Soc. 81: 129–140. tion. April 2008 Journal of Japanese Botany Vol. 83 No. 2 87

Korhonen K. 1978. Interfertility and clonal size in the of the 8th Interational Conference on Root and Butt Armillaria mellea complex. Karstenia 18: 31–42. Rots, IUFRO Working party (S2.06.01, August 9– Kusano S. 1911. Gastrodia elata and its symbiotic as- 16, 1993). pp. 376–382. Wik, Sweden and Haikko, sociation with Armillaria mellea. Imperial Univ. Finland. Tokyo, J. Coll. Agric. 4: 1–65. Tashima Y., Terashita T., Umata H. and Matsumoto Mohammed C., Guillaumin J J. and Berthelay S. 1994. M. 1978. In vitro development from seed to flower Armillaria species identified in China and Japan. in Gastrodia verrucosa under fungal symbiosis. Mycol. Res. 98: 607–613. Trans. Mycol. Soc. Jpn. 19: 449–453. Mwangi L. M., Lin D. and Hubbes M. 1989. Terashita T. 1985. Fungi inhabiting wild orchids in Identification of Kenyan Armillaria isolates by cul- Japan (III). A symbiotic experiment with tural morphology, intersterility groups occurring in Armillariella mellea and Galeola septentrionalis. British Columbia. Can. J. Microbil. 31: 651–653. Trans. Mycol. Soc. Jpn. 26: 47–53. Nagasawa E., Komatsu M. and Maekawa N. 1991. The Ministry of Health, Labor and Welfare 2006. Taxonomic reassessment of Armillaria mellea in Japanese Pharmacopoeia (15th ed.). 1243 pp. Japan. Report for a Grant-in-Aid for Scientific Tokyo. Research No. 63560155, Ministry of Education, The Pharmacopoeia Commission of People’s Republic Science and Culture of Japan. of China 2005. Pharmacopoeia of the People’s Ota Y., Matsushita N., Nagasawa E., Terashita T., Republic of China (2005). I: 39. Chemical Industry Fukuda K. and Suzuki K. 1998. Biological species Press, Beijing. of Armillaria in Japan. Plant Dis. 82: 537–543. Thomas H. E. 1934. Studies on Armillaria mellea Roll-Hansen F. 1985. The Armillaria species in (Vahl) Quel., infection, parasitism, and host resis- Europe. Eur. J. For. Path. 15: 22–31. tance. J. Agric. Res. 48: 187–218. Suzuki K., Fukuda K., Shiga Y., Matsushita N. and Togashi I. and Takizawa N., 1992. Bottle cultivation of Terashita T. 1994. Identification of Japanese bio- Armillaria species using carrot. Proc. Hokkaido logical species of Armillaria by isozyme patterns. Branch, Jpn. Wood Res. Soc. 24: 43–46. In: Johansson M. and Stenlid J. (eds.), Procedings

菊地 原a, 樋口正視a, 吉村宏昭a,諸田 隆a,鈴 木彰ｂ：オニノヤガラ (ラン科) とナラタケ (キ シメジ科) の in vitro 共生試験 感染塊茎で増殖が認められたことから, これら 2 オニノヤガラ Gastrodia elata 塊茎より分離した 種は菌根を形成し共生していることが実験的に証 ナラタケ Armillaria を子実体形成による形態的特 明された. 菌根形成が認められた塊茎は, 菌根形 長の観察および単胞子分離菌による交配試験を行 成が無い塊茎および対照区と比較し, 重量および うことにより種を同定した. その結果, 共生菌は 個体数に有意な増加が見られた. Armillaria gallica であることが明らかになった. (a株式会社ツムラ生薬本部生薬研究部, この菌を用いてオニノヤガラ塊茎と二員培養を試 b千葉大学教育学部) みたところ, 塊茎の皮層に菌糸進入が確認され,