Response Mechanisms Of Dendrobium Officinale Inoculated With Cladosporium Halotolerans And Key Environmental Factors For Its Seed Germination

Dendrobium officinale Kimura et Migo is a perennial herb of Orchidaceae and requires mycorrhizal fungi in their growth processes from the seed germination to the completion of seedling development.The dendrobe which roots are localized by mycorrhizal fungi is called mycorrhizal dendrobe.These funguses,localizing in dendrobe roots and forming mycorrhiza,are called mycorrhizal fungi of dendrobe.Root structure of D.officinale is generally divided into three parts: root plexus,cortex(including outer cortex,cortical parenchyma and endothelial layer),and middle column.Fungi invaded the epidermal cells of roots,passed through the channel cells in the outer cortex or destroyed the roots plexus into the cortex,formed pelotons in the cortical cells.This is a hallmark of the mycorrhizal formation in orchids including D.officinale.Dendrobe seedlings were inoculated with the Cladosporium halotolerans isolated before from the roots of dendrobe seedlings.During the 3-month co-cultured with the C.halotolerans,the dendrobe seedlings grew well.The seedling roots were light yellow or yellowish brown while the control group roots were white;The observation of paraffin section and mycorrhizal slide showed that C.halotolerans had invaded into the roots of dendrobe and formed clearly visible pelotons in the cortex cells.It had been proved that C.halotolerans was a beneficial symbiotic mycorrhizal fungus of dendrobe seedlings.Dendrobe seedlings inoculated with fungus were taller than control plants.The stems of mycorrhizal dendrobe were strong-thick and well-proportioned,and the leaves were bright green.All roots of the plants were embedded in the medium,with a few aerial roots;The control plants were short and weak,their stems were thin,and the apexes of basal leaves occasionally appeared yellowing.Their roots developed well and some of them grew into air forming aerial roots.Surface of the aerial roots were attached with root hair.Fresh weight of stem,leaf and plants of the dendrobe seedlings infected with fungus were significantly heavier than those of controls,reaching at 40%,100% and 75% respectively;The number of stem node,internode length,internode width and stem height of dendrobe seedlings infected with fungus increased 15%-38% compared with those of controls.The average leaf thickness of dendrobe seedlings was significantly thicker than that of control,increased 28.8%.The paraffin sections observation showed that the leaves of dendrobe were composed of upper and lower epidermis,mesophyll and veins.The thickening of the leaves was mainly due to the thickening of the mesophyll,after inoculation of the fungus.Physiological and biochemical studies showed that C.halotolerans could improve the physical and chemical properties of D.officinale seedlings.The leaf conductivity and malondialdehyde(MDA)content were significantly lower than the control.The leaf conductivity was low showed that electrolyte leakage of the leaf cells was small;The low MDA content showed that the cell membrane was less damaged.Enzyme activity of the dendrobe seedlings leaf of protective enzyme system(SOD enzyme,POD enzyme and CAT enzyme)were significantly higher than the control groups,increased 25.53%,50% and 37.19%,respectively.Polysaccharide content increased 12.5%,which improved the quality.The content of chlorophyll was higher than that of the control.Fungal hyphae of C.halotolerans helped to absorb nutrients for growth and development of dendrobe seedlings,its root activity was significantly lower than the control.Almost all of orchids require mycorrhizal fungi in their growth processes from the seed germination to the seedling development.In different stages,there were different types of mycorrhizal fungi that were required for the seedlings’ growth development.A agar block(1 mm×2 mm)of C.halotolerans fungus was inoculated near dendrobe seeds on the surface of germination mediumum.Co-cultured dynamics were observed for six weeks.After the first week,the dendrobe embryos entered the first stage of germination(the embryos swelled and turned green);After the second week,the embryos entered the second stage of germination(the embryos expanded and the seed coats ruptured);After the third week,the embryos continued to expand,but the speed of expansion began to slow down;The fourth week later,the embryos enlargement basically stopped,no longer grow,no differentiation of vanes,and stagnated in the third stage of germination(the emergence of primary meristem).C.halotolerans was not a beneficial symbiotic fungus in the process of seed germination because it could not promote the differentiation of dendrobe seeds embryonic vanes.The dendrobe seeds were tiny.The nutrients stored in the proembryo were limited,which cannot satisfy the nutrient supply during the whole germination process.It was necessary to absorb nutrients from outside to satisfy its growth and development.Some environmental factors can affect the dendrobe seeds germination.Other abiotic environment factors expect fungi had a great impact on the germination of seeds,including nutrition,light,moisture and oxygen.Controling these main abiotic environment factors,some interesting results were observed.The seeds germination required external nutrition.Without additional nutrient supply,the development of seed embryos stopped at the first stage of germination because of the trace nutrients stored in the original embryos.In the first week after sowing,the embryos expanded to green and readied for self-cultivation of photosynthesis under sufficient water and suitable temperature conditions.It was very important to provide light at this time.The consequence of dark culture could result in no vane differentiation,distort and twistion embryos,which cannot complete the whole process of subsequent germination.When seeds completely immersed in water,the embryos were excessively swelled but without cell proliferation.Seeds of dendrobe were sowed into 3mm-5mm deep in solid nutrient medium to reduce the oxygen suplying,which can accelerate the seed embryos into the fouth stage(the first vane differention)or the fifth stage(the second vane differention)of germination.C.halotolerans colonized dendrobe seedling roots to develop mycorrhiza,which can significantly improve morphological,physiological and biochemical characteristics.It was a beneficial symbiotic fungus for dendrobe seedlings growth.However,it was not a beneficial symbiotic fungus during the germination of dendrobe seeds because C.halotolerans inhibited the differentiation of vanes and could not promote the germination of seeds.Nutrition,light,moisture and oxygen are essential among the key non-biological factors affecting the dendrobe seeds germination.Although the embryos were small and stored limited nutrients,they can rapidly turn green after absorption and enlarge,and carried out photosynthesis to promote the differentiation of vanes.Adequate water was beneficial to embryo absorption and ruptured the seed coats,but only the first week needed more water,moisture content should be appropriately reduced with the growth of embryos.The decrease of oxygen content in the environment can stimulate embryos to speed up the early vane differentiation and accelerate the germination process of dendrobe.