Study On Diversity Of Orchidaceae Mycorrhizal Fungi And Its Influence On Orchid

Mycorrhiza is a symbiont which formed by plants and fungi through a long term co-evolution.The Orchidaceae is a typical mycorrhizal plant,nearly every orchid can coexist with fungi,and the symbiotic systems run through the whole life cycle of the plant,from seed germination to blossom and yield fruit.To investigate the diversity of Orchidaceae mycorrhizal fungi and its function on orchid growth is significant in orchid resources conservation,wild population recovery and industrial management.In this research,11 Cymbidium and 41 Dendrobium were selected to isolate the symbiotic fungi,and then all the isolated fungi were identified and grouped by molecular methods.And on this basis,the fungal communities' diversity among different samples and the specialization between fungi and plants were studied.Meanwhile,the symbiotic systems between different growth stages of plants(seeds,protocorms and seedlings)and fungi were established under the tissue culture condition.The fungi which had positive functions on orchid growth were picked out.Based on these results,the specificity of plants and fungi was discussed.Meanwhile,two gene families,Ca M and CML,which are related to mycorrhizal-fungi symbiosis,were analyzed using bioinformatics methods from Phalaenopsis equestris and Dendrobium officinale genomes.The main results are as follows:(1)1450 mycorrhiza fungi were isolated from 11 species of Cymbidium,these fungi were belonged to 3 Families(Tulasnellaceae,Ceratobasidiaceae and Sebacinaceae)and divided into 22 OTUs;the mycorrhiza from C.ensifolium in Sichuan province had the greatest species richness(7 OTUs),the mycorrhiza from C.faberi(Zhengjiang),C.goeringii(Guizhou)and C.goeringii(Hunan)had the lowest species richness(1 OTUs).(2)A total of 1434 mycorrhizal fungi were isolated from 41 species of Dendrobium plants and divided into 48 OTUs,these fungi were distributed in the family Tulasnellaceae,Ceratobasidiaceae and Sebacinaceae;the species richness was the highest among Dendrobium guangxiense(7 OTUs),the species richness is the lowest,Dendrobium nobile,Dendrobium nobile,and Dendrobium nobile(only 1 OTU).(3)One-way analysis of variance(ANOVA)and non-parametric multivariate analysis of variance(PERMANOVA)revealed differences in the community structure of mycorrhizal fungi among these samples.For the 11 species of orchids used in this study,it is speculated that there may be a specific relationship with mycorrhizal fungi at the plant species level,geographical distribution level,growth environment level,growth and development level;while 35 selected Dendrobium plants may have a specific relationship with mycorrhizal fungi at the plant species level and geographical distribution level.(4)The fungi of Tulasnellaceae were more suitable to establish a symbiotic relationship with Orchidaceae on the concentration of 4.0 g/L OMA;the Tcs1 strain could establish a symbiotic relationship with all the tested orchids at all oatmeal concentrations;The fungi of Ceratobasidiaceae was more suitable to establish a symbiotic relationship with Orchidaceae on concentration of 0.25 g/L OMA,and it was more suitable for Sebacinaceae to establish a symbiotic relationship with Orchidaceae in a concentration of 12.0 g/L OMA.(5)Four mycorrhizal fungi with the ability to promote the Orchideace seeds germination were screened out,and their nutrient medium for symbiosis culture were also confirmed.Among the 4 fungi,Paphiopedilum corrugatum can only intergrowth with strain Tcs1.(6)Six mycorrhizal fungi that promoted the growth and development of the rhizomes(protocorms)of Orchidaceae were screened out.After 30 days of co-cultivation with Tcs1 strains,the net increase rate of average fresh weight was up to 203.47%(2.0 g/L OMA),170.65%(8.0 g/L OMA)and 168.55%(8.0 g/L OMA)respectively for the Cymbidium mannii,,Cymbidium ensifolium and Dendrobium officinale.(7)Six mycorrhizal fungi that promoted the growth of Orchid seedlings were selected.After 30 days of co-cultivation with Tcs1 strains,the net fresh weight gain rates of Cymbidium mannii,Cymbidium ensifolium and Dendrobium officinale reach as high as 179.01%(2.0 g/L OMA),158.84%(2.0 g/L OMA)and 167.51%(4.0 g/L OMA)respectively,and colonization rate were 100%;Strain Tof4 had a certain specific relationship with D.candidum seedlings on 8.0 g/L OMA.(8)Bioinformatics methods were used to analyze the Ca M and CML gene family of Phalaenopsis equestris and Dendrobium officinale.4 Ca M proteins and 54 CML proteins were predicted in the whole genome of Phalaenopsis equestris and Dendrobium officinale;phylogenetic analysis divided them into 10 subfamilies;9 genes in Phalaenopsis equestris were in the leaves.The expression of the two genes was up-regulated relative to flower,stem and root.The expression of the two genes in the leaves was down-regulated relative to the flowers,stems,and roots;the expression of the four genes in the symbiotic germinal library of Dendrobium.officinale was relatively non-symbiotic.The library was upregulated and the expression of the 4 genes in the symbiotic germinal library was down-regulated relative to the non-symbiotic library.This indicates that the Ca M and CML gene family may be involved in the biological regulation of seed germination of Dendrobium officinale and Phalaenopsis equestris symbiotic germination.