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 aBotanicalRawMaterialsResearchDepartment,BotanicalRawMaterialsDivision, Tsumura&Co.,3586,Yoshiwara,Ami-machi,Ibaraki,300-1192JAPAN; ＊E-mail: [email protected] bFacultyofEducation,ChibaUniversity,Chiba,263-8522JAPAN (RecievedonAugust20,2007) An Armillaria species isolated from Gastrodia elata was identified as A. gallica based on morphological characteristics of basidiomata and mating test. Symbiosis be- tweenG.elataandA.gallicawasconfirmedbyusingtwo-membercultures.Hyphalcoils ofthefungalsymbiontwereformedinthecorticalcellsoftubersandisolatedfromtheir corticalcells.Lengthandweightoffungus-infectedtubersweresignificantlyhigherthan 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. In order 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; to investigate the vegetative reproduc- (1911)andHamada(1940).ChaandIgarashi 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 —77— 78 植物研究雑誌 第83巻 第2号 平成20年4月 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 symbiontandthelattercollectedin2001was 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 thesestructurestoPetridishescontainingpo- 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,on4June1995. sawdust medium (beech sawdust: rice bran 3 : 1 ; 60(cid:1)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- Table1. Mycorrhizaformationandrhizomorphsadhetionto Gastrodiatubers Tubersinthefield Tubers invitro Rhizomorphs observed mycorrhizal observed mycorrhizal Adhesion 19 9 26 19 Nonadhesion 104 0 0 0 Total 123 9 26 19 April2008 JournalofJapaneseBotanyVol.83No.2 79 Table2. Haploidtestersof ArmillariaspeciesandtheiroriginsinJapan Biologicalspecies Hosttree Locality A.gallica Abiesveitchii Mt.Simagareyama,NaganoPref. A.veitchii Mt.Simagareyama,NaganoPref. A.cepistipes Cryptomeriajaponica MogamiTown,YamagataPref. C.japonica MogamiTown,YamagataPref. A.ostoyae Chamaecyparisobtusa SiwaTown,IwatePref. C.obtusa IwateTown,IwatePref. A.tabescens Quercusacutissima AmiTown,IbarakiPref. Q.acutissima AmiTown,IbarakiPref. 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(cid:1)0.5ºC for 40 days in the dark. batedat25.0(cid:1)0.5ºCfor6monthsuntilfungal For fruiting induction, the bottles were sub- rhizomorphs were formed actively. Eight jected to 16.0(cid:1)1.0ºC under intermittent illu- cultures were made in this way. minationwith350lxalternatedwiththedark 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(cid:1)0.5ºC for 3 months in Mating test thedark.Asthecontrol,eighttuberswerein- 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- Matingexperimentswereperformedbyplac- tioned and observed under a dissecting mi- ing inocula 1 mm apart on a 1.25(cid:1)malt ex- croscope and scanning electron microscope tract agar and incubated at 25(cid:1)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(cid:1)0.5ºC for 2 weeks on haploid tester strains of several Armillaria PDA. species.Thetesterstrainswerefirstpairedin 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 Hosttreesoffungalsymbiontwerenotde- 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 darkbrownishcrustosecoloniesonPDAme- 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 Identification of Armillaria species Gastrodia elata. Daughter tubers grow around a decayedmothertuberlocatedundertheflowering 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=2cm. April2008 JournalofJapaneseBotanyVol.83No.2 81 Fig. 4. Daughter tubers attached and penetrated by rhizomorphs extended fromthemothertuber.Bar=4mm. Fig. 5. Basidiomata of Armillaria gallica. The basidioma was formed after three weeksincubationunderthelight. 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 Table3. Matingtestofhaploidisolatesbetweenmycorrhizal Armillariatesters Testerspeciesandtheirsexualtype Mycorrhizal symbiont A.gallica A.cepistipes A.ostoyae A.tabescens (Sexualtype) 1 2 1 2 1 2 1 2 1 + + – – – – – – 2 + + – – – – – – 3 + ? – – – – – – 4 + ? – – – – – – +:Compatiblepairing; –:Incompatiblepairing; ?:Uncertainpairing. Fig.6. GastrodiaelatatuberculturedwithArmillariagallicaonthesawdust medium for three months. Budding tubers were observed on the inocu- latedtuber(it)adheredbythetherhizomorphs.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 showninthetotallengthoforiginalandbud- 4). ding new tubers. Mycorrhizal tubers pro- There were significant differences April2008 JournalofJapaneseBotanyVol.83No.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=2cm. Table4. Sizeof Gastrodiaelata tubersincubated with Armillariagallica forthreemonths Fungaltreatment Numberoftestedtuber Length(mm) Freshmass(g) Inoculated 26 75.8＊(cid:1)(3.6) 0.8＊(cid:1)(0.05) Control 8 10.0 (cid:1)(1.8) 0.2 (cid:1)(0.04) ＊:SignificantdifferencebetweeninoculatedtuberandcontrolP<0.05.Numberinparen- thesesshowstandarderror Fig. 8. Mycorrhiza formed on Gastrodia elata tuber. rz. Rhizomorphs. m. Mycorrhiza.p.Parenchyma.ep.Epidermis.Bar=500 (cid:2)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 (cid:1)m. between mycorrhizal and non-mycorrhizal tubers.Thereforeadhesionofrhizomorphsto 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 mycorrhizalandnon-mycorrhizaltubersand 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 hyphalcoilsofA.gallica.Ontheotherhand, 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 April2008 JournalofJapaneseBotanyVol.83No.2 85 Fig.10. Lengthof Gastrodiatubersincubatedwith Armillariagallica. ＊P< 0.05vs.nonmycorrhizaltubers;+P<0.05vs.control. Fig.11. Weightof Gastrodiatubersincubatedwith Armillariagallica. ＊P< 0.05vs.nonmycorrhizaltubers;+P<0.05vs.control. mothertuberextendedtodaughtertubersand 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- weakpathogenofconiferforestsandlivedto gestions. decay dead hardwoods and litter, on the other hand, A. ostoyae is a strong pathogen References and lived to decay healthy coniferous trees. AndersonJ.B.andUllrichR.C.1979.Biologicalspe- Ota et al. (1998) showed A. gallica would be cies of Armillaria mellea in North America. a weak pathogen and is found most com- Mycologia71:402–414. ChaJ.Y.andIgarashiT.1995.Armillariaspeciesas- monly in the field, while A. ostoyae and A. sociatedwithGastrodiaelatainJapan.Eur.J.For. melleaseemtobestrongpathogensinJapan. Path.25:319–326. The association between Gastrodia elata ChaJ.Y.,SungJ.M.andIgarashiT.1992.Biological and Armillaria species was reported by species and morphological characteristics of Kusano (1911) and Hamada (1940), how- Armillaria mellea complex in Hokkaido: A. ostoyae and A. bulbosa. Res. Bull. Exp. For. ever, they did not identify the Armillaria at HokkaidoUniv. 49:185–194. specific level. Cha and Igarashi (1995) re- ChaJ.Y.,SungJ.M.andIgarashiT.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.Mycoscience35:39–47. ChaJ.Y.,SungJ.M.andIgarashiT.1995.Armillaria Dessureault, and A. singula Cha & Igarashi. mellea(Vahl:Fr.)Kummers.s.fromHokkaido.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., RishbethJ.,TermorshuizenA.J.,TirróA.andVan (Cha and Igarashi 1995). 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. Ein Nachtrag zur Mykorrhiza von Galeola species produced abundant rhizomorphs in septentrionalisReichb.f.Jpn.J.Bot.10:387–463. the soil and was a weak pathogen. These Hamada M. 1958. Mykotrophische Symbiose. Trans. featuresprobablyhavetheadvantagetoform 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 TokyonougyousyoinTokyo(inJapanese). Kile G. A. and Watling R. 1983. Armillaria species is formed with the balance of continuously from south-eastern Australia. Trans. Br. Mycol. formed mycorrhiza and mycorrhizae diges- Soc.81:129–140. tion.