Studies On Endangerous Wild Orchids And Its Relationship With Mycorrhizal Fungi In Yunnan Province

Orchids is refer to the species in Orchidaceae family. Chinese orchid is classified as genus Cymbidium in Chinese traditional concept. In most cases, people refer Chinese orchid to terrestrial orchids in genus Cymbidium in orchudaceae family. The orchids have high valve in ornament and economy and they are very diverse and widely distributed in Yunnan province. At present, development and utilization of orchids resource are in a primary stage. Because demand of orchids is much higher than its supply, the farmers driven by economic benefit started to excavate wild terrestrial orchids on the mountain .The natural resources of terrestrial of orchids were destroyed seriously. If the method of large quantity propagation of orchid is not developed, protection of terrestrial of orchids will be kept in talking. Currently, the tissue culture was used. But the tissue culture plantlets have very low survival rate after transplanting, growth slowly, delayed blossom or no blossom without mycorrhization. It was found that Endophytic fungi of orchid can provide more nutrients, hence promote growth and development through mycorrhization .Therefore, mycorrhization in tissue culture is a useful technique for the large quantity propagation of endangerous Orchids. To understand the relationship between orchids and endophytic fungus, fungi strains from natural roots of orchids were isolated and inoculated into the roots of Cymbidium goeringii Rchb.f. C.hookerianum and Dendrobium devonianum (growing in moss mix in with vermiculite)and Cymbidium goeringii Rchb.f. under tissue culture and soil culture condition. Through inoculation under tissue culture condition, some good strains that can promote the growth of orchids significantly were screened. By using indicators such as fresh weight increment of growth, micro-and ultra-micro-structure of cortical cell of the roots, re-isolation and inoculation, molecular biology, etc, it is confirmed that a symbioses culture system for Rhizoctonia sp. and orchids was established. By the observation under electronic microscopy, the process that the hyphae was dissolved and absorbed by orchids root and other change in ultra-micro structure were observed. The research is aimed to establish a methods for propagation of orchids plantlet by tissue culture with inoculation of endophytic fungi. We believe this method maybe useful for propagation of other rare orchid species.. The detail results are shown as follows: 1 . The seeds of Cymbidium Goeringii, C. hookerianum, Dendronbium devoninum can be induced to germinate and form seedlings on culture media. Adding some coconut milk and specific orchids nutrition solution into medium provided necessary nutrients and environmental for orchids seed development. Meanwhile the maturities of several orchids were correlated with the time of germination. The high the maturity was, the shorter the time of germination was, vice versa. Other factors influenced germination time were active carbon which can prevent the brownization and vitamin C. The best media in Cymbidium Goeringii were selected as follows: The media 1/2MS +NAA 0.5mg/L +6-BA 3.0mg/L was the best for protocorm induction; 1/2MS+6-BA 1.0 mg/L+NAA 0.05mg/L was the best for adventitious bud induction; 1/2MS+6-BA 2.0 mg/L +NAA 0.1mg/L was the best for adventitious bud propagation (propagation coefficient was 4); 1/2MS+ NAA 0.5 mg/L was the best for rooting of adventitious buds. 2. 184 representative fungi strains were isolated from the 89 orchids seedlings from 8 main sites of orchids in Yunnan. Among those strains, 64 strains showed a high frequency of infection or had been reported as endophytic mycorrhiza fungi. They are classified as 18 groups according to colonial morpha,hyphal characteristic,fruiting organ,spore morpha and derivation fruiting body as follows: Rhizoctonia sp., Orchidaceous rhizotonias, Chaetomium sp. ,Trichoderma sp.,Fusarium sp.,Diplococcoium sp. Acremonium sp.,Sarcinella sp., Papulaspora sp.,Trichosporiella sp.,Gliomastix murorum,Mycena,Cafenularia piceae,other genus of Hyphomycetes. 3 . CLB111, CLB113 (from wild Cymbidium goeringii Rchb.f.) and Kw214(from wild Cattley) is proved to be a good endophytic fungi strains for Cymbidium goeringii Rchb.f. C. hookerianum and Dendrobium devonianum respectively through inoculation, biomass measurement, re-isolation, DNA detection and Micro and ultra-micro-observation of chip of root. The three trains mentioned above were inoculated into the roots of plantlets of Cymbidium goeringii,C. hookerianum. After 4.5 months of symbioses culture, plantlets showed a normal leave color, well developed root system. The fresh biomass of Cymbidium goeringii inoculated with CLB111, CLB113and KW214 increased by 70.6%, 70.2% and 66.1% respectively while that of control is 45.6%. Statistic showed that the difference between that of inoculation and control was significant (that of CLB111, CLB113 were extremely different from that of control (a=0.01) ). Inoculated with MLX102 ( isolated from C. sinense in Xishuangbanla)and CLN114( isolated from Cymbidium goeringii in Nujiang prefecture)also showed a significant increase of growth in C. hookerianum and Dendrobium devoninum plantlets. From the fact that tissue culture plantlets ofCymbidium goeringii,C. hookerianum,Dendrobium devoninum inoculated with different strains had developed mycorrhizal and had better growth appearance, we may infer that the same species of orchid may be infected by different strains of mycorrhizal fungi if it grows in different places and different species may be infected by same strains of mycorrhizal fungi if they grow in the same place. This is proved again that mycorrhizal fungi do not have strong specificity. 4. The nutrients content in the roots of tissue culture plantlet (no mycorrhizal), symbioses mycorrhizal and mycorrhizal of wild seedling in Cymbidium goeringii were analyzed and the result showed: N, P, K contents in symbioses mycorrhizal is lower than that of roots of tissue culture plantlet (no mycorrhizal), but slightly higher than that of wild mycorrhizal. The N content in roots of tissue culture plantlet (no mycorrhizal) is as much as 2.0 times that of wild mycorrhizal and 1.5 times that of symbioses mycorrhizal; The P contents in roots of tissue culture plantlet (no mycorrhizal) is as much as 2.5 times that of wild mycorrhizal and 1.5 times that of symbioses mycorrhizal; The K contents in roots of tissue culture plantlet (no mycorrhizal) is as much as 5.0 times that of wild mycorrhizal and 3.0 times that of symbioses mycorrhizal; This means that roots of Cymbidium goeringii can directly absorb the nutrient in the man-made culture nutrition solution and accumulate them in the roots, while wild orchids usually grow in the sandy soil under the shrub cover where soil humus layer is thin, nutrient supply is not enough, and cause the lower nutrients accumulation in the roots. 5. The DNA marker detection with RAPD and re-isolation of strains proved that a symbioses culture system with Rhizoctonia sp.and seeds of Cymbidium goeringii ,C. hookerianum and Dendrobium devoninum. Electrophoretic patterns of fungi strains CLB111 Cymbidium goeringii and C. hookerianum has two special fragment that is 2000 bp ,1500 bp and Electrophoretic patterns of fungi strains CLB113 C.goeringii has three special fragment that is 1500 bp,1031 bp 和700 bp. Electrophoretic patterns of 1500 bp is common fragment of of fungi strains CLB111 and CLB113. 6. The observation under optical and electronic microscope for CLB111,CLB113 and KW214 found the pathway of infection and structure of mycorrhizal in Cymbidium goeringii,C. sinense,C. hookerianum and Dendrobium devoninum. For the terrestrial orchids, the infection pathway is that fungi invaded velamen firstly , then entered cortical tissue by breaking the velamen. But for Dendrobium devoninum of epicole orchid, the fungi enter the cortical tissues through passage cell, and the colonized and infected other cells continuously by means of hyphaepenetrating the cell wall. There are a lot of pelotons which appear in dark color and in the irregular shapes in the cortical tissue under microscope observation. Under the electronic observation, it was found that pelotons is a cell nuclear surrounding by a group of hyphae. Pelotons mainly appear in outsider layer cells of the cortical tissue and it will provide the nutrients for plants when it is dissolved and absorbed. With more invasion and growth of young hyphae endophytic fungi can provide nutrition for rapid growth of the plantlets.