Study On Ectomycorrhizal Effect And Symbiosis Mechanism Of Castanea Henryi

Chinquapin(Castanea henryi)is one of the most important woody grain species in southern China,which plays an important role in building beautiful countryside,poverty alleviation and green growth in mountainous areas.In the process of afforestation in the poor and arid hills,the survival rate of seedlings was low,long slow seedling period and high artificial management cost.Therefore,it is important to solve urgently that how to cultivate high-quality strong seedlings of chinquapin to adapt to poor mountainous areas.Mycorrhizal seedling afforestation can significantly improve the survival rate,stress resistance and plant growth.Chinquapin is the host of ectomycorrhizal(ECM)fungi.To explore the physiological effects of ECM on seedlings and its symbiotic mechanism is the basis of the application of mycorrhiza seedlings which has important practical meaning and scientific value.Thus,this study using C.henryi and Boletus edulis as the materials constructed ECM symbiont.Firstly,to clarify its mycorrhizal structure characteristics.Secondly,to study the effects of ECM of C.henryi on seedling physiology and nutrition,and to clarify the mycorrhizal effect and seasonal characteristics in the field.Thirdly,analyze the genes related to ECM formation and physiological effects of C.henryi,clarify their expression level characteristics,and then analyze the mycorrhizal effect and symbiosis mechanism of C.henryi.Among these,the tissue culture technology,and the methods of cell anatomy and physiological and biochemical,combined with technics of high-throughput sequencing,bioinformatics and qRT-PCR have been used.Aiming at providing a theoretical for the application of the mycorrhizal biotechnology for chinquapin.The main results are showed as follows:(1)Established symbiosis system in field and in vitro.The ECM symbiosis system in field was established by C.henryi seedlings and B.edulis soild fungicide,the symbiotic matrix was the selected loess:peat:perlite:vermiculite=4:1:1:1(v/v/v/v)mixed matrix.After six months latter,the infection rate was up to 60%in field.The ECM symbiosis system in vitro was established by the sterile seedlings,which obtained from stem segments of the seedlings of C.henryi after rooting induction(MS+1.5 mg/L IBA,rooting rate reached 76.70%),and B.edulis liquid fungicide,the symbiotic matrix was the selected peat:sand=3:1(v/v)sterilization matrix.The hyphae began to infect the roots from the fourth week,and the infection rate continued to rise,the infection rate at the 24^thweek was 43%,and the infection rate did not increase significantly after that.Four months later in field and two months in vitro,the anatomical structure of the root system was observed by stereomicroscopy,paraffin section and laser confocal,and it was found that the root tips formed hyphae mantle(12?47.97?m)and harting net(the fungal cells invaded into the epidermal intercellular space of the 1-2 layers of the root tip and stopped at the cortical cells),and the finger like tips of harting net full with a number of rough endoplasmic reticum and mitochondria,which confirmed that a functional symbiosis was established.(2)Effects of C.henryi ectomycorrhizal on seedlings physiology and nutritionField investigation and sampling were carried out,which found that B.edulis inoculation in field could significantly improve the photosynthetic physiology,promote the uptake of soil nutrients(N,especially P),and increase the biomass of seedlings(especially the biomass of underground part).(P<0.05).The Pn(10.26 mol·m^-2·s^-1)of JG was 1.8times higher than that of CK.The total biomass of JG(26.73 g/seedling)was 1.8 times that of CK,and the biomass of underground part of JG(14.60 g/seedling)was 2.15 times that of CK.The total N content(168.03mg/seedling and 155.95mg/seedling)of the overground and underground parts of JG is 1.2 times and 1.3 times of that of CK,respectively.The total P content(60.80mg/seedling and 96.50mg/seedling)in the overground and underground parts of JG was 2.7 times and 2.8 times higher than CK,respectively,and the activity of soil acid phosphatase in JG was 17.29 times higher than CK.The cell anatomical structure of ECM in field was studied by field investigation combined with scanning electron microscopy and paraffin section technology in different seasons.The results showed that,in autumn,the mycorrhizal surface mycelium was seen to be fluffy and dynamic,and the normal mantle and harting net were observed.In winter,the mycelium on the mycorrhiza surface adhered and flaked,and the mycelia invaded into the epidermal cells and the epidermal cells were suberized,mantle turned more thinner,and mycelium into the epidermal cells.In spring,a large number of mycorrhizal and rhizomorph were observed,and most of the mycorrhizal were germinating new lateral roots.In summer,the mycelia on the surface of the ectomycorrhizal were slightly agglutinated.Plant physiological and biochemical methods were used to determine the mycorrhizal tips(JG),non-mycorrhizal tips(WXC)and uninoculted seedlings root tips(CK)of C.henryi in different seasons.The results showed that,compared to autum and spring,involved in cold resistance metabolism of SOD,POD and CAT activity increased in winter,and the content of MDA and soluble sugar increased,soluble protein and PAL activity were reduced,at the same time,the root activity was also significantly lower than that in autumn and spring.However,no matter in what season,the antioxidant capacity and root activity of mycorrhizal tips were significantly higher than inoculated seedlings with WXC and CK,while there was no significant difference between WXC and CK.(3)Molecular mechanisms of ECM symbiosis formation and its effects.According to the infection rate and fresh weight of seedlingsin vitro,the mycorrhizal formation and effects were the most significant at 24 weeks after inoculated.Therefore,the transcriptome libraries of ECM roots and non-ECM roots at this time were established.The transcriptome data were analyzed,and a total of 4,353 differentially expressed genes(DGEs)were obtained(P<0.05,and|log₂FC|?1),of which 2,615 were up-regulated and1,738 were down-regulated.The metabolic pathways of DEGs related to ECM formation including 77 MARK signaling pathways,60 plant hormone signal transduction pathways,35 plant and pathogen interactions,10 endocytosis pathways,9 flavonoid biosynthesis pathways and 7 sesquterpenoids biosynthesis pathways.Homologous genes associated with arbuscule mycorrhizal and nodule formation including 17 mitogen-activated protein kinases,3 symbiotic receptor kinases Sym RK/NORK,2 cytokinin receptor kinases LHK and 5 nucleoprotein NUP.The metabolic pathways and genes of DEGs related to light absorption,carbon fixation and sugar export were:11 photosynthesis pathways,6photosynthesis antenna proteins,15 Calvin cycle pathways,23 starch and sucrose metabolism pathways,and 3 SWEET genes related to sugar export of host plants.qRT-PCR analysis of 2 DEGs(ENOD and ERF1)and 4 DEGs(rbc S,RPL35A,WAXY and SWEET),which related to ECM formation and physiological effects,respectively.The results showed that the expression of these genes increased significantly in ECM roots,which was consistent with the results of RNA-seq.