Studies On Mycorrhizal Biology Of Dendrobium

Shihu is a rare traditonnal Chinese medicine which includes many species of medicinal plants in the genera of Dendrobium,and also there are many kinds of Shihu herbs for commercial use in China.Shihu's identification usually was only by its structural characteristics of stem,and that will not be insufficient for the classification of the close species.So,it is very important to find root morphological characteristics to meet the identification need of different plants in Dendrobium.We are all known that the plants as Shihu are becoming extinct because of the need increasing of Shihu herbs,and the special habitat of this kind of plants as well as its low propagate rate,and the difficults to cultivate Dendrobium plants under artificial conditions.Dendrobium plants have mycorrhizal characteristics of Orchidaceae,which means that the plans must be formed mycorrhiza with symbiotic fungi in all their life cycle from seed germination,development of seedlings.The anatomical characteristics of roots and orchid mycorrhiza of 34 species of plants in Dendrobium,which included the isolation of endophytic fungi,its evaluation to promoting growth effectives on D.nobile and D.candidum,and identifications of effective fungi,were systematically studied in this thesisThe root structures of Dendrobium were typical structures of monocotyledons,which was consisted of velamina,exodermis,cortex,endodermis,pericycle,vascular cylinder and pith from outside to inside.The V-thickened cells in innermost layer of velamen,tilosomes,type of thickened wall in exodermis,shape of ceil in endodermis and ratio of cortex diameter and stele radius could be used as specific character for the identification of the species Dendrobium species.Since the number of layers in velamina was easy to be influenced by the growing environment,it might not be properly to be used as a character for phylogeny identification in Dendrobium.The shapes of velamina are correlated to the epiphytic habit of Dendrobium roots.Velamina are frequently narrower on the side of a root attached to a substrate than that on the exposed side.Comparative anatomy between the wild Dendrobium and the seedlings showed that number of layers in velamina, diameter of velamina and number of vascular cylinder in Dendrobium seedlings are less than that in the wild one.Phylogenetic assessment of anatomical features data showed that some species are cluster in a dendrogram,which was congruent with Dendrobium section.This results was not consistent with the phylogenetic cladogram of the rDNA ITS sequence data.Moreover,the molecular cladogram of molecular data was inconsistent with the classification of section in Dendrobium genera.The anatomical characters of orchid mycorrhiza were observed with the light microscope.The hyphae penetrated into the velamina from the outmost cells or root hair cells and existed in the intercellular or intracellular of velamina.The hyphae colonized in the intracellular space cell and formed hyphal coil in velamina of D.nobile.The root exodermis of Dendrobium was comprised of a series of thick-walled dead cells and few thin-walled passage cells at irregular intervals.The hyphae in velaman cells only penetrated the thin-walled passage cells and formed hyphal peloton structure surrounding the nucleus.Once across the exodermis layer through the passage cells,the mycorrhizal fungal hyphae entered the thin-walled cortical cells and formed typical orchid mycorrhizal structure(peloton) therein.The peloton included the vigorous peloton and the digested one.Two types of peloton could co-exist in one cortex cell.Distribution of peloton in the cortex was related with the epiphytic habit of Dendrobium roots.Pelotons were frequent distribution on the side of a root attached to a substrate than on the exposed side.In cortex,peloton mainly colonized the middle layers cells and hardly existed in innermost 1～2 layer cells. Mycorrhizal fungi usually could not infect the endodermis,pericycle and vascular cylinder.But we observed the infected passage cells in the endodermis and pericycle of D.candidum and D. wardianium.Colonized ratio of the peloton in cortex is from 0 to 80%,but D.williamsonii is the special one with a ratio of 70～80%,D.longicornu doesn't form the peloton in cortex.Comparative anatomy of roots between the wild Dendrobium and the cultivated seedlings showed that colonized ratio of peloton in cortex of the wild Dendrobium is more than that of the cultivated seedlings.The digested pelotons existed only in the cortex of the weak cultivated seedling.Pure fungal cultures were successfully obtained using tissue isolation method.1313 endophytic strains were isolated from 43 samples of Dendrobium.Fungal colonization rate and fungal isolation rate are different in each Dendrobium plant.The endophytie fungi existed in Dendrobium plants have the character of diversity,and the coefficient of diversity was from 1.75 to 4.33.Based on the morphological characteristics,these fungi were divided into ten distinct morphotypes.The relative frequency of these morphotypes was different in each species of Dendrobium.Interaction between each fungus and seedlings was studied by co-culture.36 strains had significant positive effects on the growth of seedlings of D.nobile in various growth parameters including fresh weight,dry weight,stem diameter,number of new roots,length of longest root, number of new buds,as well as height of highest bud.And 8 strains inoculated to D.candidum seedlings greatly enhanced seedlings' plant height,stem diameter,new roots number and biomass. Anatomical features of the inoculated roots with different fungi were observed.Each inoculated fungi could infect the velamina of seedlings.However,some of which could penetrate the cortex cells through passage cells.Fungi 22-R-4 and G08-R-17 could form the pelotons in the cortex cells respectively.The fungi with effectives for growth promotion of plants could not infect the endodermis and vascular bundle sheath,but which was exception for other fungi with harmful to seedlings.Endophytic fungi inoculated to seedlings could affect the content of chemical compositions of plants.The isolate 22-R-4 not only could promote the growth of D.nobile and D. candidum,but also enhanced the content of alkaloid for the first plant and polysaccharide for the second one on MS co-culture medium.The isolate 22-R-4 inoculated to D.nobile seedlings also affected the content and distribution of auxin in the seedlings.Combined with classic morphologic classification,Saccardo and Hughes-Tabaki-Barron fungal classification system,17 of 36 effective strains were identified which were subjected to 14 genera. Ten species of fungi without conidiophore were identified by sequencing the PCR-amplified rDNA ITS1-5.8S-ITS2(internal transcribed spacer ITS) regions.In comparison with similar taxa,the sequence was grouped together with the known strains.