| As an important plant resources, the Orchidaceae not only has extremely economic and ornamental value, but also has special value for medicinal and food. However, the wild resources are under the threat of endangerment day by day due to over-collection and habitats destruction. Although the protection aroused many peoples’attention, human damage and the huge demand for wild resources have not been eased, the conservation work was difficult and task. Therefore, based on endophytic fungi of Orchidaceae conservation research has an important significance on the maintenance of species diversity and the industrial development of orchid. We isolated endophytic fungi from the roots of wild Dendrobium catenatum Lindley.which collected from Hunan province. By inoculating with seedlings of D. catenatum in vitro to screening beneficial endophytic fungi, and to further identifitied. Simultaneously, combined with three beneficial endophytic fungi from D. loddigesii to study symbiotic seed germination of D. catenatum, the physiological characteristics of endophytic fungi on the protocorms and seedlings. The result investigated the specificity between endophytic fungi and D. catenatum, and the growth-promoting mechanism in D. catenatum. The main results were as follows:(1) Thirty seven endophytic fungi strains were isolated from the roots of wild D. catenatum by slice separation. The best sterilize time is 3 min. Strains from PDA and FIM medium were different. By inoculating with seedlings of D. catenatum in vitro,9 strains of endophytic fungi were screened with the positive effect on the growth. According to morphological characters and ribosomal DNA(rDNA) sequence analysis, two strains were belong to Epulorhiza sp.(the identities were 100% and 96%), one strain was classed to Guignardia camelliae(the identity was 100%), one was classed to Mycosphaerella cruenta(the identity was 99%), and the position of other five growth-promoting fungi in classification systems were incertae. However, they were closely related to GenBank codes EU482263, GQ996178. AM901831、GU566294、GU566256, the identities were 96%、95%、99%、98% and 100%, respectively.(2) Seven strains from D. catenatum (C20、C22、C35、C42) and D. loddigessi (L12、L24b and L28) were used in symbiotic seeds germination of D. catenatum in vitro. After 100 days cultivation, strains C22、C35 and C42 caused seeds death, but strains C20、L12、L24b and L28 promoted seed germination, but to various degrees. Specifically, seeds co-cultured with strains L24b and L12 had a remarkably significantly higher germination rates (88.48% and 76.04%, respectively) than that of the control(63.23%), and had a extremely significantly to promote seeds developing seedlings; Strains C20 and L28 had a significant effect on the development of protocorms.(3) Seven endophytic fungi mentioned above were inoculated with the protocorms of D. catenatum indicated that strains C35、C42、L12 and L28 caused protocorms death, but strain C22 increased the growth rate of fresh weight remarkably. The content of N in protocorms which inoculated with strains C20、C22 and L24b were significantly higher than control, and the content of K in protocorms were significantly higher than control, strains C22 and C20 increased the content of P significantly; Strains C22 promoted the content of chlorophyll a and b obviously. The endogenous hormones of protocorms co-cultured with C22、C20, and control were measured, the result showed that both two strains increase GA3 concentration extremely significantly. Therefore, strains C22 and C20 both played a remarkable role in morphogenesis and physiological characteristics.(4) Seedlings of D. catenatum co-cultured with seven endophytic fungi mentioned above. The average growth rate of fresh weight and the content of chlorophyll a,b in treatments were higher than in control, but the average growth rate of fresh weight in seedlings inoculated with strains C42、C35、C22 had evident difference, the content of chlorophyll a,b in strain C42 had significant difference, strains C20、C42、C35 and C22 could increase the content of N、P、K, contrast with control. Seedlings co-cultured with C42、C35、C22 and C20, and control were used to study the effects of fungi on the endogenous hormones, the result showed that the content of GA3 in inoculated seedlings had been extremely significantly increased, strains C20、C35 and C42 promoted IAA concentration markedly significantly, but the ABA concentration in inoculated seedlings were obviously lower than control. Therefore, strains C20、C22、C35 and C42 would be applied in cultivation and production of D. catenatum.(5) Seven endophytic fungi were inoculated with the seeds, protocorms and seedlings of D. catenatum showed that no one fungus was beneficial to all three key stages simultaneously. D. catenatum required different fungi in different stages, and had two or more beneficial fungi species in every stage, possed a wider range of fungi. These findings suggested that D. catenatum have a low degree of mycobiont specificity under in vitro.(6) The endophytic fungi on the physiological characteristic of protocorms and seedlings indicated that endophytic fungi regulate growth and development of D. catenatum by endogenous hormones, which GA3 played an important role. In addition, endophytic fungi could promote plant to uptake of nutrients and improve the capacity of photosynthesis, thus contributed to plant growth and development. |
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