Rhizoctonia Fungi Enhance the Growth of the Endangered Orchid Cymbidium Goeringii
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20 Rhizoctonia fungi enhance the growth of the endangered orchid Cymbidium goeringii Jianrong Wu, Huancheng Ma, Mei Lu¨ , Sufen Han, Youyong Zhu, Hui Jin, Junfeng Liang, Li Liu, and Jianping Xu Abstract: Orchids are among the most prized ornamental plants in many societies throughout the world. As a result, con- sumer demands have created a significant pressure on wild populations of many species, including Cymbidium goeringii Rchb. f., a rare terrestrial orchid endemic in China, Korea, and Japan. To help conserve natural populations of C. goerin- gii, we recently started investigating methods to cultivate these orchids. Here we fulfilled Koch’s postulates and demon- strated that fungal strains isolated from the roots of natural Cymbidium plants increased fresh mass, plant height, number of leaves, and root length of C. goeringii, and that the two fungal strains originally isolated from C. goeringii showed overall greater effects on growth than two other strains from other Cymbidium species. Internal transcribed spacer se- quence analyses revealed that the four fungal strains likely represented at least two new taxonomic groups, both belonging to the family Ceratobasidiaceae of the Rhizoctonia fungi. Our study demonstrated that these fungal strains could potentially help the commercial cultivation of the increasingly rare and endangered orchid C. goeringii. Key words: orchids, mycorrhizal fungi, symbiosis, molecular marker. Re´sume´ : A` travers le monde, les orchide´es se retrouvent parmi les plantes ornementales les plus appre´cie´es. Conse´quem- ment, la demande par les consommateurs a cre´e´ une pression significative sur les populations naturelles de plusieurs espe`- ces, incluant le Cymbodium goeringii Rchb f., une orchide´e ende´mique rare, en Chine, Core´e et Japon. Afin d’aider a` la conservation des populations naturelles du C. goeringii, les auteurs ont re´cemment de´marre´ une recherche pour permettre la culture artificielle de ces orchide´es. Ils ont boucle´ le postulat de Koch et de´montre´ que des isolats fongiques obtenus de racines naturelles de plants de Cymbidium stimulent la croissance du C. goeringii pour l’ensemble des caracte`res examine´s (poids frais, hauteur des plants, nombre de feuilles et longueur des racines) et que les deux souches fongiques obtenues originalement du C. goeringii montrent une plus forte stimulation ge´ne´rale que les deux autres souches provenant de d’au- tres espe`ces de Cymbidium. Les analyses de se´quences ITS re´ve`lent que les quatre souches fongiques proviennent vraisem- blablement d’au moins deux nouveaux groupes taxonomiques, les deux appartenant a` la famille des Ceratobasidiaceae du complexe fongique Rhizoctonia. Cette e´tude de´montre que ces souches fongiques pourraient aider la production commer- ciale du C. goeringii menace´, et de plus en plus rare. Mots-cle´s:orchide´es, champignons mycorhiziens, symbiose, marqueur mole´culaire. [Traduit par la Re´daction] Introduction phytic orchids from mountainous regions of China, Korea, The Orchidaceae is the world’s largest plant family and Japan, Southeast Asia, and India (DuPuy and Cribb 1988; contains over 25 000 species (Arditti 1992; Berg-Pana Llamas 2003). Their beautiful flowers and their ability to 2005). Of the 1000 or so genera in Orchidaceae, the genus survive and grow in cold and temperate environments have Cymbidium, or ‘‘boat orchids’’, is among the best known made them among the most sought-after ornamental plants and most widely grown of all orchids and orchid hybrids that have long attracted horticultural enthusiasts. Indeed, (DuPuy and Cribb 1988; Berg-Pana 2005). Cymbidium in- Cymbidium orchids have been cultivated for thousands of cludes over 50 species of terrestrial, lithophytic, and epi- years, starting in China and gradually expanding to other Received 24 June 2009. Published on the NRC Research Press Web site at botany.nrc.ca on 22 January 2010. J. Wu. Key Laboratory of Forest Disaster Warning and Control, Southwest Forestry University, Kunming 650224, China; Key Laboratory of Biodiversity Conservation in Southwest China, State Forestry Administration, Southwest Forestry University, Kunming 650224, China. H. Ma and M. Lu¨. Key Laboratory of Biodiversity Conservation in Southwest China, State Forestry Administration, Southwest Forestry University, Kunming 650224, China. S. Han and H. Jin. College of Forest Resources and Environment, Nanjing Forestry University, Nanjing, 210037, China. Y. Zhu. Physiopathology Laboratory, Yunnan Agricultural University, Kunming 650201, China. J. Liang1 and J. Xu.2 Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON L8S 4K1, Canada. L. Liu. Key Laboratory of Forest Disaster Warning and Control, Southwest Forestry University, Kunming 650224, China. 1Present address: Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, 510520, China. 2Corresponding author (e-mail: [email protected]). Botany 88: 20–29 (2010) doi:10.1139/B09-092 Published by NRC Research Press Wu et al. 21 parts of the world, including Europe and North America. gal strains isolated from the roots of natural Cymbidium The popularity of Cymbidium in North America is reflected plants should enhance the growth of C. goeringii. The sec- by a society and an annual conference specifically dedicated ond is that fungi isolated from C. goeringii roots should to this genus (www.cymbidium.org). show greater enhancements in C. goeringii plants than those At present, most of the >50 species in the genus Cymbi- isolated from other Cymbidium orchids. The phylogenetic dium can be found in the wild, and limited artificial propa- affiliations of the four strains within the broad Rhizoctonia gation is available for most species within this genus, alliances were analyzed and discussed. including the terrestrial orchid Cymbidium goeringii Rchb. f. However, artificial propagation is generally slow Materials and methods and cannot meet the consumer demand. Cymbidium goerin- Seeds and seedlings gii is native to southwestern and northeastern China, the Ko- rean peninsula, and Japan (Chung and Chung 2000; Lee The ripe seed capsules of Cymbidium goeringii were col- 2002). This orchid is among the most highly prized orna- lected in a natural forest in the Baoshan region, Yunnan mental plants in the world, with some plants sold for thou- Province, southwestern China. The dominant trees in this sands of US dollars apiece (Light 2007). In recent years, forest were Quercus acutissima Carr., a broad-leaf evergreen the consumer demand for orchids, especially for C. goerin- species, and Pinus yunnanensis Franch., a coniferous tree gii, has increased dramatically both within and outside of endemic to Yunnan. The sampled site was at 1980 m above China, for private homes, businesses, and large-scale public sea level, with an average annual temperature about 16 8C, displays. The increasing demands have put significant pres- and a range between 10 and 30 8C. To identify an appropri- sures on natural populations of C. goeringii, especially in ate medium to germinate C. goeringii seeds, we tested a to- southwestern China (Anonymous 2004). How best to meet tal of 48 media formulations. The starting culture medium the consumer demand and conserve the natural populations was that established for germinating seeds of other orchids of this and other orchids are two significant challenges fac- by Liu et al. (2002) and Xu and Guo (1989). The medium ing conservation biologists and horticulturists. contained the half-strength Murashige and Skoog (MS) basal One approach to meet the challenges described above is medium (Murashige and Skoog 1962; product M5519, to develop effective cultivation methods. Several methods Sigma-Aldrich, Oakville, Ontario, Canada). However, we to germinate orchid seeds have been established (e.g., further experimented with a range of concentrations for Thompson 1977; Liu et al. 2002). However, plantlets of ter- three ingredients: a-naphthalene acetic acid (NAA: 0.2, 0.3, restrial orchids from germinated seeds typically have very 0.4, and 0.5 mg/L), coconut milk (10%, 15%, 20%), and ac- low survival rates after transplanting, grow slowly, with de- tivated carbon (2, 3, 4, and 5 g/L). Seeds were incubated at layed or no flowering (e.g., Liu et al. 2002; Shimura and 25 8C with a 12 h light – 12 h dark cycle. The medium with Koda 2005; Batty et al. 2006; Stewart and Kane 2006). In 1/2MS + 0.5 mg/L NAA +15% (v/v) coconut milk + 3 g of contrast, orchid seeds inoculated with mycorrhizal fungi activated carbon/L was found most suitable for germinating have shown enhanced germination and greater growth than C. goeringii seeds, yielding 96% germination rate for the those not inoculated with any fungi (e.g., Peterson et al. ripe seeds. 1998; Dearnaley 2007; Otero et al. 2007). Indeed, the im- portance of mycorrhiza in orchid seed germination was first Orchid collection and fungal strain isolation discovered about a hundred years ago by a French botanist, To obtain natural mycorrhizal fungal strains from Cymbi- Noel Bernard (Bernard 1909). Arditti (1984) provided a de- dium plants for testing, we collected four Cymbidium plants tailed account of the historical research activities associated representing three species native to Yunnan province. Two with the roles of fungi in orchid seed germination. In addi- C. goeringii plants were collected from the Baoshan region tion, it has been known since the 1930s that mycorrhizal in southwestern Yunnan; one Cymbidium sinense plant from fungi are