| The Orchidaceae are a family of typical mycorrhizal plants.Mycorrhizal symbiotic fungi have been proven to be a key limiting factor in their cultivation,introduction and domestication,resource regeneration and conservation.Epiphytic orchids account for about 69%of the orchid species.Compared with that of terrestrial orchids,deep research of mycorrhizal symbiosis of epiphytic orchids was relatively deficient,and many aspects were to be explored.Dendrobium is the second largest group of epiphytic orchids with ornamental and medicinal value.The conservation and sustainable use of its resources have become the focus of universal attention.Dendrobium sinense Tang et Wang,an endemic and endangered species in Hainan Island,is distributed only in a small number of mountainous areas in the midwest.The study of mycorrhizal biology of D.sinense is the prerequisite of using its symbiotic fungi resources,which is of great significance in maintaining species diversity,horticulture cultivation and medicinal production.There is still a lack of systematic study on its mycorrhizal biology.Therefore,this study chose to carry out the mycorrhizal biology of D.sinense from several aspects including the diversity of mycorrhizal and endophytic fungi,the morphology and structure mycorrhizal roots,in vitro symbiotic seed germination,the establishment of symbiotic system and the physiological response to drought stress.This study attempted to discuss the diversity,the specificity and internal mechanism of mycorrhizal symbiosis of D.sinense.The results may help to understand the nature of mycorrhizal symbiosis of D.sinense,and provide the reference for future conservation and sustainable utilization of epiphytic orchids.The research results and innovative content show as the following:1.The diversity of mycorrhizal and endophytic fungi of D.sinenseThis study explored the associations between mycorrhizal and endophytic fungi isolated from the roots of D.sinense and four host tree species,which were two preferential host tree species,Syzygium buxifolium and Rhododendron moulmainense,and two non-preferential host tree species,Podocarpus neriifolius and Cyclobalanopsis disciformis.(1)There were 421 endophytic fungal isolates obtained from 36 host trees.56 fungal operational taxonomic units(OTUs)were identified.71 Rhizoctonia-like isolates(7 OTUs)all belonged to Tulasnellaceae.Species of Xylariaceae(25 OTUs,RF=27.79%)were the most dominant fungal endophytes isolated.Trichoderma,Tolypocladium,Xylaria,Pestalotiopsis were the dominant fungal genera.(2)The species richness and diversity of mycorrhizal and endophytic fungal communities were strongly influenced by host tree species.Both species richness and diversity indices showed that D.sinense roots on S.buxifolium harbored the most diverse and abundant endophytic fungi.(3)The analysis of community composition indicated that endophytic fungal species of Tulasnellaceae were dominant on S.buxifolium and R.moulmainense but infrequent on C.disciformis and P.neriifolius.(4)The results indicated that the diversity of mycorrhizal and endophytic fungi may reflect the orchid-host preference,and provided evidence for distinct mycorrhizal and endophytic fungal communities on different host tree species.2.The morphology and structure of the mycorrhizal roots of D.sinenseThis study compared the wild and mycorrhizal cultured seedlings of D.sinense.The morphological and anatomical characteristics,fungal invasion way,distribution and digestion-growth of mycorrhizal roots were observed by paraffin sections and optical microscopy.(1)The cross section of D.sinense root was composed of the velamen,the cortex and the pith,and the exodermis was distributed with passage cells.There were no significant differences in the basic structure of wild and cultured roots,and the key difference was whether there were invasive fungi in the roots.(2)The mycorrhiza of D.sinense was typical as an epiphytic orchid,and mycorrhizal fungi invaded cortical cells through passage cells.The continuous invasion and digestion of the hyphae provided the nutrition,and three different forms of mycelium including the hyphae,pelotons and peloton pieces were observed.(3)7 Tulasnellaceae strains were all able to establish a symbiotic relationship with D.sinense,which were true mycorrhizal fungi.(4)The total colonization rate of mycorrhizal fungi,the colonization rate of the hyphae and pelotons of mycorrhizal cultured roots were significantly higher than that of wild mycorrhizal roots,but the colonization rate of peloton pieces was significantly lower.The results indicated that mycorrhizal fungi were more susceptible to colonize cultured roots.3.In vitro symbiotic germination of D.sinense seedsThis study chose wild D.sinense seeds to carry out in vitro symbiotic germination on OMA medium with 7 Tulasnellaceae strains,which were certified as true mycorrhizal fungi of D.sinense.(1)After 16 weeks of sewing,the asymbiotic D.sinense seeds remained at stage 1-2.All 7 tested strains could successfully infect D.sinense seeds,and the seed germination was promoted to varying degrees.(2)All tested strains except Tul-07 could promote the germination process of seeds entering stage 3 or above.Seeds treated by Tul-06 were always in the higher stage,and seeds treated by Tul-01 and Tul-04 entered stage 4,and only seeds treated by Tul-06 and Tul-03 entered stage 5.(3)The analysis of Total Germination Rale,Germination Rale Index and Growth Index all showed that Tulasnella sp.Tul-06 was the most significant strain on seed germination and protocorm development of D.sinense,Tul-03 was the second,and Tul-07 was the worst.(4)The results indicated that under in vitro conditions,D.sinense seeds and the mycorrhizal fungi could establish symbiotic relationship,and there was no strict specificity between them.There was also no stage specificity since that Tul-06 can both significantly promote the seed germination and the further development of the protocorm.4.The establishment of endophytes-seedlings symbiotic system of D.sinenseThis study established the symbiotic system between cultured D.sinense seedlings and mycorrhizal endophytic fungi,and screened promoting fungal strains and "fungi +bacteria" combination by comparing the effects on symbiotic seedlings of mixed inoculation with endophytic bacteria.(1)Modified DE medium was a more suitable symbiotic medium,because the symbiotic relationship between cultured seedlings and mycorrhizal endophytic fungi was more harmonious.(2)Based on indicators such as the fresh weight,tillers and roots,8 promoting fungal strains were selected,including Tulasnellaceae sp.Tul-02,Tul-05,Tul-07,Fusarium sp.Fus-01,Theleporus sp.The-01,Trichoderma sp.Tri-01,Tri-02,Tri-03.Among them,Tul-07 and Tri-01 could promote the seedlings growth extremely significantly.(3)The antagonism screening was performed bythe tablet confrontation culture method between 8 promoting endophytic fungal strains and 3 promoting endophytic bacteria strains.10 non-antagonistic combinations were selected from 24.After symbiotic culture,4 promoting combinations including Tul-05+Bac-01,Tul-07+Bac-01,Fus-01+Ser-01,Tri-01+Bac-01 were screened out.(4)The best promotingcombinations Tul-07+Bac-01 and Tri-01+Bac-01 could promote the absorption of mineral elements(N,P,K,B,Fe,Mn),improve photosynthetic performance(chlorophyll a and b,net photosynthesis rate and stomatal conductance)and regulate endogenous hormones(IAA,GA3,ABA),and showed synergistic effect on some indexes,respectively.(5)The results indicated that co-inoculation with endophytic fungi and bacteria could be feasible to promote orchid growth.Bac-01(Bacillus weihenstephanensis)may be the mycorrhiza helper bacteria,and showed no strict specificity for fungi.5.The physiological response of symbiotic D.sinense seedlings to drought stressThis study chose PEG to simulate drought stress,the changes of plant morphology and the differences of related physiological indexes of different inoculated D.sinense seedlings were observed and analyzed.(1)Under the condition of PEG simulated drought stress,the morphological and structural changes of cultured D.sinense roots were observed,including chlorophyll reduction,rhizoids production,2-3 layers of velamen cells increased.(2)The photosynthetic performance indexes(chlorophyll,net photosynthesis rate and stomatal conductance),the content of osmotic regulating substances(proline,soluble sugar,soluble protein)and the activity of antioxidases(SOD,POD,CAT)were positively correlated with the drought resistance of D.sinense seedlings,and the membrane permeability indexes(plasmalemma permeability,MDA)was negatively correlated with the drought resistance.(3)The results indicated that drought stress can lead to adaptive changes of D.sinense roots,inhibit the photosynthetic performance,damage the membrane permeability,increase the osmotic regulation and antioxidases activity of D.sinense seedlings,and the longer the stress,the more obvious the effect.(4)Single inoculated treatments Tul-07 and Tri-01 could only significantly increase the fresh weight of symbiotic seedlings,which could not significantly improve the drought resistance.The dual combinations Tul-07+Bac-01 and Tri-01+Bac-01 could significantly improve the drought resistance.(5)The analysis of related physiological indicators showed that the two combinations Tul-07+Bac-01 and Tri-01+Bac-01 could relieve the inhibition of photosynthetic characteristics,reduce the membrane permeability,and increase the accumulation of osmotic regulating substances and the activity of antioxidases of D.sinense seedlings under drought stress to varying degrees,respectively.The results indicated that the potential mycorrhiza helper bacteria Bac-01(B.weihenstephanensis)could assist mycorrhizal endophytic fungi to improve the drought resistance of D.sinense seedlings. |
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